Surface Immobilization of Viruses and Nanoparticles Elucidates Early

Subscriber access provided by University of South Dakota

Article

Surface Immobilization of Viruses and Nanoparticles Elucidates Early Events in Clathrin-mediated Endocytosis Marta Fratini, Tina Wiegand, Charlotta Funaya, Zhongxiang Jiang, Pranav N.M. Shah, Joachim Spatz, Elisabetta A Cavalcanti-Adam, and Steeve Boulant ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00134 • Publication Date (Web): 10 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

Surface Immobilization of Viruses and Nanoparticles Elucidates Early Events in Clathrin-mediated Endocytosis Marta Fratini1,2, Tina Wiegand2, Charlotta Funaya3, Zhongxiang Jiang4, Pranav N.M. Shah1,5, Joachim P. Spatz2, Elisabetta Ada Cavalcanti-Adam2,#,*, Steeve Boulant1,2,#,* # Equal contribution * Address correspondence [email protected]

to:

[email protected]

or

1Heidelberg

University, Department of Infectious Diseases, Virology and German Cancer Research Center, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany 2 Max

Planck Institute for Medical Research, Department of Cellular Biophysics, Jahnstrasse 29, 69120 Heidelberg & Heidelberg University, Institute of Physical Chemistry, Department of Biophysical Chemistry, Im Neuenheimer Feld 253, 69120, Heidelberg, Germany 3

Heidelberg University, Electron Microscopy Core Facility, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany 4 Leica

Microsystems GmbH, Am Friedensplatz 3, 68165 Mannheim, Germany

5

Present address: Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts, United States of America

ACS Paragon Plus Environment

1

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 49

Clathrin-mediated endocytosis (CME) is an important entry pathway for viruses. Here, we applied click chemistry to covalently immobilize reovirus on surfaces to study CME during early host-pathogen interactions. To uncouple chemical and physical properties of viruses and determine their impact on CME initiation, we used the same strategy to covalently immobilize nanoparticles of different sizes. Using fluorescence live microscopy and electron microscopy, we confirmed that clathrin recruitment depends on particle size and discovered that the maturation into clathrin-coated vesicles (CCVs) is independent from cargo internalization. Surprisingly, we found that the final size of CCVs appears to be imprinted on the clathrin coat at early stages of cargo-cell interactions. Our approach has allowed us to unravel novel aspects of early interactions between viruses and the clathrin machinery that influence late stages of CME and CCVs formation. This method can be easily and broadly applied to the field of nanotechnology, endocytosis and virology.

KEYWORDS: viruses, nanoparticles, endocytosis, clathrin-mediated endocytosis, membrane curvature, click chemistry


2

Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Clathrin-mediated endocytosis (CME) is a central process that relocates membrane, membrane-associated receptors and extracellular cargo inside the cell to regulate multiple cellular functions and maintain cell homeostasis1. CME starts with the recruitment of adaptor proteins at the plasma membrane (PM), which promote clathrin assembly and lead to the nucleation of a clathrin coated pit (CCP)2,3,4. Engagement of further adaptor/accessory proteins and curvature effectors coordinates the growth of CCPs5. Finally, the recruitment of the GTPase dynamin marks the scission of the newly formed vesicle, leading to the release of a clathrincoated vesicle (CCV) into the cytosol6. Although many proteins have been identified as potential CME regulators, the spatial and temporal determinants that control and coordinate the initiation and formation of CCPs at the PM are poorly understood7,8. Viruses are strict intracellular parasites as they need to gain entry into the cell to replicate. CME constitutes an important entry pathway for many viruses 9. While typical virus size ranges from 40 to 100 nm (e.g. Dengue virus, reovirus), some viruses can be as small as 20-30 nm (e.g. parvovirus, norovirus) or even larger than 300 nm (e.g. vesicular stomatitis virus, herpesvirus, Megavirales). The size and shape of viruses directly influence the cellular mechanisms leading to their clathrin-dependent internalization10,11; however, the early factors determining clathrin machinery recruitment and CCPs maturation upon virus attachment, are not yet clear 12. Analogous to viruses, colloidal nanoparticles have been shown to be internalized through different cellular pathways in a size-dependent manner10,11. For spherical nanoparticles, Rejman J et al.13 related particle size and internalization pathways in the absence of specific cell receptor interactions: particles smaller than 200 nm in diameter are generally internalized by CME while particles larger than 500 nm enter cells via caveolae-mediated structures13. For polystyrene particles in particular, a size of 100 nm is more efficiently internalized by CME in comparison to 50 or >200 nm. Interestingly, for gold nanoparticles, the preferred diameter for clathrin


3


Page 4 of 49

internalization is 50 nm14. For both viruses and nanoparticles, the molecular and biophysical determinants driving these size-dependent entry mechanisms are still uncertain. This gap of knowledge is mainly related to the lack of a method to study virus-cell early interactions, which are highly unpredictable events that take place in a extremely narrow time-window (1-2 minutes)10,15. Most of the studies on particle uptake are performed using free particles/viruses suspended into the cell media10,11,13,14 , a condition that makes it difficult to deeply investigate how a cargo might favor the activation of the cellular endocytic machinery. Additionally, using free particles makes it technically very difficult to look at how cargo alters the biochemical and physical characteristics of an endocytic structure. It was recently shown that local membrane curvature imposed by the presence of external objects (e.g. nanocones and nanopillars) can induce recruitment of Bar proteins leading to the formation of CCPs16,17. As such, it is possible that extracellular cues induced by viruses and nanoparticles act as determinants for the sizedependent endocytic mechanisms by regulating early stages of CME and CCP commitment. In this work, we propose a method to spatially and temporally control virus and nanoparticle interactions with the clathrin-dependent endocytic machinery at the single particle level. By using surface chemistry to immobilize virus particles on coverslips we were able to spatially control endocytic events providing us with a methodological approach allowing us to better study the interaction between cargo and the endocytic machinery. This methodology alleviates the limitations of using free particles suspended in culture media. By using fluorescence microscopy and electron microscopy we were able to address how molecular and external mechanical stimuli contribute to size-dependent endocytosis. We investigated how early interactions between virus/nanoparticles and the clathrin machinery influence recruitment, maturation and CCP commitment during CME.


4

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION

Virus capsid bio-conjugation does not hinder viral particle uptake and preserves virus infectivity To exert a spatio-temporal control over the recruitment of the CME machinery onto viruses and nanoparticles, we chemically modified the surfaces of viruses and nanoparticles to immobilize them on substrates. This allowed us to characterize early steps of cargo/endocytic machinery interactions with the immobilized particles. Chemical modification of virus capsids represents a research field in constant growth. In the last years, several approaches have been described to achieve chemical bio- conjugation18,19,20,21 of the virus coat. Here, we used a click chemistry based approach to modify and covalently immobilize reovirus, a well characterized non-enveloped virus of approximately 80 nm in diameter, which enters cells through CME2,15. This method represents an innovative approach to study early virus-cell interactions, since it circumvents the major drawbacks of the canonical assays that employ “free” virus particles, namely: the high spatial motility and transient interaction of viruses at the cell surface, and the fast internalization rate. Briefly, the outer capsid proteins of the virus were chemically modified by targeting the free amine functional groups for the conjugation of a propargyl-N-hydroxysuccinimidyl ester linker (alkyne-linker) and Alexa647-succinimidyl ester (Figure 1A). The alkyne linker serves as a functional group for the copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction to achieve the covalent immobilization of viruses on the substrate (Figure 1A). The fluorescent dye allows the visualization (Figure S1A) and tracking of single virus particles to specifically monitor their interaction with the clathrin endocytic machinery through live-cell confocal microscopy2,15. To rule out the possibility of particle aggregation due to the addition of chemical


5


Page 6 of 49

groups on the virus surface, virus particles labeled with Alexa647 dye (Alexa647-virus) or labeled with both Alexa647 and Alkyne-linker (Alexa647/Alkyne-virus) were deposited on glass. Particles were imaged by fluorescence microscopy and the fluorescence intensity of individual virus particles was quantified (Figure S1A). For both types of modification, virus particles showed similar distribution of fluorescence intensity characteristic for fluorescentlylabeled virus particles10,15. Such a distribution is compatible with a variable number of fluorophore molecules per virus particle and indicates that the functionalization strategy did not induce particle aggregation. Scanning electron microscopy of virus particles also demonstrated that the addition of the fluorophore and alkyne-linker did not induce particle aggregation (Figure S1B). Alexa647/Alkyne-viruses have an average diameter of 79 nm ± 16 nm, which was similar to the average diameter of non-modified viruses (77 nm ± 14 nm) (Figure S1B). These findings were further corroborated by dynamic light scattering analysis of functionalized and nonfunctionalized virus particles (Figure S1C). Viruses are proteinaceous particles that cannot be modified under chemical conditions without the risk of losing their infectivity due to structural alterations. To determine whether our functionalization strategy affected virus infectivity, non-modified and Alexa647/Alkyne-viruses were titrated in reovirus permissive and replication competent BSC1 cells22,23. Cells were infected with serial dilutions of virus particles and, 16-18 h post infection, cells were fixed and immunostained against the reovirus non-structural protein NS, which is a marker for reovirus replication in the cytosol of infected cells 22. Quantification of virus infectivity revealed that functionalization of virus particles had no impact on their infectivity (Figure S2A). To control that virus replication was not affected by chemical modification, BSC1 cells were infected with non-modified virus and Alexa647/Alkyne-viruses using an identical Multiplicity of Infection (MOI=1). Virus progeny were collected three days post infection, from the supernatant of


6

Page 7 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


infected cells and titred in BSC1 cells. Immunofluorescence staining of the reovirus nonstructural protein NS confirmed that the replication efficiency of Alexa647/Alkyne-viruses was not affected compared to non-modified viruses (Figure S2B). We concluded that our functionalization of the free amine functions on the surface of virus capsids does not induce particle aggregation and has no effect on virus infectivity and replication efficiency.

Viruses are covalently linked to substrates via click chemistry Our strategy to covalently link virus particles onto a glass surface is illustrated in Figure 1A. Glass coverslips were modified by formation of a self-assembled monolayer consisting of silane-PEG(3000)-Azide (Si-PEG-N3). The bio-conjugation of Alexa647/Alkyne-viruses to the azide groups on the surface was performed by a CuAAC click reaction. To control the specificity of virus particle immobilization, similar concentrations of Alexa647-virus and Alexa647/Alkynevirus were clicked onto Si-PEG-N3 coverslips in absence or presence of the copper catalyst. The number of immobilized virus particles and their fluorescence intensity was determined by confocal microscopy (Figure 1B). In the absence of alkyne moieties on the virus particles and in the absence of the copper catalyzer for Alexa647/alkyne-viruses, very few particles were observed on the glass substrate by fluorescence microscopy. On the contrary, efficient immobilization was observed when Alexa647/alkyne-viruses were linked by a CuAAC click reaction to the surface (Figure 1B). The experimental conditions were adjusted to obtain an optimal virus distribution on glass coverslips for subsequent imaging (approximately 8.0 ± 0.6 particles/ 10 m2). Importantly, the immobilized particles showed similar fluorescence intensity distributions compared to Alexa647/Alkyne-virus deposited on glass coverslips (non-covalently


7


Page 8 of 49

bound to glass surface) (Figure 1C), indicating that the virus particles did not form aggregates during the copper-catalyzed covalent immobilization onto the surface. To make the glass surfaces competent for cell adhesion, the cRGDfK-alkyne peptide (cyclo[ArgGly-Asp-D-Phe-Lys]), was also immobilized by a CuAAC click reaction24. This peptide serves as a ligand for integrins and promotes the formation of focal adhesions25, which are large cell adhesion structures tethering cells to surfaces. Cells seeded on Si-PEG-N3 coated coverslips or on Si-PEG-N3 coated coverslips with clicked reovirus only were not able to attach and spread on surfaces. On the contrary, cell adhesion and spread occurred when cRGDfK peptides were covalently linked prior to the seeding of cells (Figure S3). Cells seeded on clicked cRGDfK only or on coverslips displaying both the cRGDfK peptide and clicked reoviruses showed no difference in seeding efficiency and focal adhesion formation. To fully control that our Alexa647/Alkyne-virus were covalently immobilized on glass surfaces, we seeded cells on viruses coated coverslips and tested whether cells could successfully uptake virus particles, which would ultimately lead to viral infection. As a control, cells were seeded on glass coverslips and infected with free (non-immobilized) Alexa647/Alkyne-viruses preincubated with click-chemistry reagents (see materials and methods). Infected cells were observed only when viruses were added to the cell culture media, whereas cells adhering onto virus coated glass coverslips remained uninfected (Figure 1D). Importantly, even 24 hours post seeding of cells on virus coated coverslips, no virus particles were observed within the cytoplasm (Figure 1D, panel 2). This observation further validated the successful covalent immobilization of viral particles at the glass surface. Taken together, these results demonstrate that our approach successfully leads to covalent immobilization of viral particles at the glass surface without altering virus infectivity and that this chemistry is compatible with cell attachment to such surfaces.


8

Page 9 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


One previous study has successfully reported the immobilization of virus particles onto glass surfaces20. However, the methodology used required genetic modifications, which are not applicable for viruses for which a reverse genetic system is unavailable20. Our approach avoids the need for genetic modifications, thus allowing for a more accessible and applicable experimental setup.

Surface immobilized viruses efficiently recruit the clathrin endocytic machinery Having established that immobilized viral particles cannot be internalized by cells, we next addressed whether these viruses retain the capacity to mediate the recruitment of the clathrin-dependent endocytic machinery. U373 cells stably expressing the  subunit of clathrin adaptor protein 2 fused to the green fluorescent protein (AP2-GFP) were seeded on coverslips coated with clicked viruses. U373 cells are a human astrocyte derived line, which are susceptible to reovirus infection26 and have been extensively used to study CME27,. AP2 is involved in coupling clathrin molecules to the PM and the fusion protein between the 2 subunit of AP2 and GFP represents a well characterized specific marker to follow live endocytic processes 2,28. By performing live-cell microscopy on U373 AP2-GFP cells seeded onto clicked-virus coated coverslips, approximately 15% of the particles located under the cell co-localized with the endocytic machinery (Figure 2A). This efficiency of endocytic machinery recruitment to the virus particles was similar to the efficiency observed when reovirus particles were added in the cell culture media2,15. Reovirus is known to use multiple pathways such as caveolae-dependent endocytosis as well macropinocytosis to enter cells 15,29. Thus, it is possible that the presence of a fraction of virus particles that do not co-localize with AP2 reflects the fact that some immobilized virus particles might be engaging other uptake pathways.


9


Page 10 of 49

We next addressed the dynamics of the clathrin-dependent endocytic machinery recruitment to immobilized viral particles. Fluorescence intensity profiles from CCPs are characterized by an intensity signal that grows over time, reaching a peak value followed by a rapid loss of fluorescence. This corresponds to the growth of a CCP, the release of the coated structure from the PM, and the uncoating of the mature CCV into the cell cytosol (Figure 2B, panel 2). The lifetime of such an endocytic event is on average a minute2,30,31 Temporal analysis of fluorescence intensity profiles for all viruses co-localizing with AP2-GFP, showed multiple cycles of AP2 recruitment, which we interpreted as recurrent formation of endocytic structures. We believe this phenotype represents multiple attempts from the cell to take up the viral particles (Figure 2B, upper panel and movie 1). As expected, fluorescence intensity profiles over time from CCPs not co-localizing with immobilized virus particles, mostly showed a single recruitment cycle (Figure 2B, lower panel). To determine whether the recurrent assembly of the clathrin machinery at the immobilized viruses is specific and induced by the presence of the particles, we measured the number of AP2-GFP recruitments co-localizing with the virus particles (referred to as “virus spots”); this value was then compared to the number of AP2-GFP recruitment events observed for CCPs that did not co-localize with clicked virus (referred as “empty pits”) and from randomly collected spots above the cell membrane (referred as “random spots”). To measure the dynamics of the AP2-GFP signal at a precise spot during a given period of time, we developed a Gaussian fitting approach allowing the automatic and unbiased characterization of the fluorescence signals over time (Figure S4A-C and details in method section). According to this method, Gaussian curves were fitted into each AP2 recruitment event upon “virus spots” and “empty pits” in order to characterize the dynamics of fluorescence recruitment. Gaussian distributions were confirmed to better fit our data set compared to other functions (e.g. Lorenzian; Gaussian with variable width) (data not shown). For


10

Page 11 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AP2-GFP recruitment on virus spots, an average of 8.1 ± 1.9 events every 10 min was observed (Figure 2C and S4D). Importantly, these values were significantly higher than the ones observed for “empty pits” (an average of 2.3 ± 2.3 events every 10 min) (Figure 2C and S4D) and random spots, which as expected displayed a random distribution (an average of 4.7 ± 4.6 events every 10 min) (Figure 2C). Detailed analysis revealed that the rate of peak occurrence on virus spots (lag time between two cycles of AP2-GFP recruitment) was 74.8 ± 37.8 s (Figure 2D, upper panel). Moreover, CCPs growing above the immobilized virus particles displayed an average lifetime of 58.96 ± 21.5 s (Figure 2D, lower panel), which is similar to the lifetime of CCPs previously reported2,3,10,15,32. To validate that the recurrent recruitment of AP2-GFP to virus spots corresponded to the recurrent maturation of CCPs and to exclude any potential artifacts deriving from protein over expression, we used genome edited cell lines expressing both clathrin fused to RFP (CLC-RFP) and dynamin fused to GFP (Dyn-GFP). Dynamin is a GTPase favors the release of the CCPs into a CCVs, and its recruitment often marks the end of an endocytic event6. By monitoring the recruitment of clathrin molecules we could identify the same recurrent behavior as in AP2 overexpressing cells (Figure S5). Furthermore, we found that dynamin was recurrently recruited to immobilized virus particles marking the end of each clathrin recurrent recruitment. This result strongly suggested that CCVs are recurrently released on top of immobilized virus particles (Figure S5). The role of cargo in inducing pit nucleation, growth and maturation during CME has been so far a matter of debate2,4,33,34. It has been shown that cargo molecules are involved in favoring CCPs maturation into CCVs2,33 as well as in inducing CCPs initiation4,34. Our data show the recurrent recruitment of the clathrin machinery and the release of CCVs above the immobilized viruses. This suggests that the presence of cargo induces the recruitment/initiation


11


Page 12 of 49

of clathrin and, most importantly, strongly suggests that the commitment to form CCVs is independent of cargo internalization, as clicked-viruses are not internalized. Taken together these results demonstrate that our system can be used to study early viruscell interaction: clicked reoviruses are capable of interacting with the cell surface and they specifically induce a recurrent recruitment of the clathrin machinery. Interestingly, the presence of a “clicked-cargo” that can actively interact with cell membrane without being internalized allowed us to unravel new details on the role of cargo in CCPs maturation and scission, which could not be addressed with previous cell biology methods.

Recruitment and assembly of clathrin-mediated structures on covalently immobilized reoviruses is independent of JAM-A receptor binding To further investigate the recurrent recruitment of the clathrin machinery on the immobilized viruses, we next determined whether reovirus cell receptors might be involved in the early steps of CME. Reoviruses enter cells through receptor-mediated endocytosis, using integrin 1 and JAM-A as genuine receptors35,36. Interference with integrin 1 partially inhibits reovirus internalization and infection of HeLa cells36,37. On the contrary, blocking JAM-A receptor binding by pre-treating cells with anti-JAM–A antibody, abolishes reovirus entry in these cells35,36. Therefore, JAM-A is a critical receptor for reovirus infection. To address whether the observed recurrent recruitment of the clathrin machinery on virus particles was receptor dependent, we blocked reovirus-receptor binding using neutralizing antibodies against JAM-A35. To validate that the anti-JAM-A antibody efficiently interferes with reovirus infection, nonmodified virus particles were used to infect HeLa cells in the absence or presence of anti-JAM-A antibody. At 16-18 hours post-infection, cells were immunostained against NS. As it was


12

Page 13 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


previously shown35,36, our results confirmed that interference with reovirus binding to JAM-A, leads to a dramatic loss of reovirus infectivity in HeLa cells (Figure 3A). Next, we performed live-cell microscopy of HeLa cells pre-incubated with anti-JAM-A antibody and seeded on virusclicked coated coverslips. Here we used HeLa cells transiently expressing clathrin light chain A; clathrin and AP2 were previously validated as co-localizing together2,3,10 at the immobilized virus spots (data not shown). Surprisingly, blocking JAM-A receptor by using neutralizing antibodies only induced a small reduction in the number of virus particles co-localizing with clathrin (52.9 ± 12.2 % of viruses co-localized with clathrin machinery in control cells, 31.6 ± 10.1 % of viruses co-localized with clathrin machinery in anti-JAM-A antibody treated cells) (Figure 3B and 3C, top panel). Nevertheless, the number of clathrin recruitment events colocalizing with virus particles was similar in untreated cells compared to cells treated with the JAM-A neutralizing antibody (control cells, 5.1 ± 1.2 recruitment events in 5 min movie duration; JAM-A antibody treated cells, 5.5 ± 1.1 recruitment events in 5 min movie duration) (Figure 3D and E). To ensure that the neutralizing antibody truly inhibits virus-JAM-A interactions, by live-cell microscopy, free virus particles were added on cells immediately after imaging. Addition of free virus particles following imaging resulted in an efficient infection of the untreated cells (Figure 3C, bottom panel, control cells), whereas cells pre-treated with the JAM-A antibody were no longer permissive to virus infection (Figure 3C, bottom panel). Taken together these results strongly suggest that the recurrent recruitment of clathrin on immobilized viruses is JAM-A receptor independent.

Surface immobilization of nanoparticles induces a size-dependent recurrent recruitment of clathrin-mediated structures


13


Page 14 of 49

We next wanted to address whether the physical properties of the viral particles could influence both the recruitment of the clathrin machinery and the commitment of CCPs to form CCVs. To uncouple the potential functions of receptor binding from the physical properties of the particles, and to determine the role of particle size on the recruitment of the endocytic machinery, we immobilized amine-modified polystyrene beads of size ranging from 20 to 1000 nm on functionalized glass surfaces. Such nanoparticles have been reported to be internalized by cells in the absence of specific interactions with cellular receptors38. The size range for internalization by CME is in the range of 50-200 nm diameter13,39,40 whereas, for diameters larger than 500 nm nanoparticles are described to be internalized by caveolae-dependent internalization pathways13. For this reason, we choose 20, 100, 300 and 1000 nm beads as representative sizes to investigate size dependency of CME recruitment. For the immobilization of the beads, we employed the same CuAAC click chemistry approach that we have used to immobilize the virus particles (Figure 1A). Observation of the clicked-beads using scanning electron microscopy demonstrated that clicked chemistry-based immobilization did not affect bead size (Figure S6). We next addressed whether the beads could induce the recruitment of the clathrin endocytic machinery. U373 AP2-GFP cells were seeded onto glass coverslips decorated with the clickedbeads and live-cell microscopy was performed. It is important to note that, during live imaging, coverslips coated with 20 nm beads often showed a high degree of aggregation; this could be explained by the higher surface/volume ratio of 20 nm objects that might favor interaction of beads. Importantly, very little aggregation was observed on 100 and 300 nm beads coated coverslips (data not shown); for this reason, image analysis was performed only on diffractionlimited objects for the 100 nm beads and on single beads for the 300 nm samples. Approximately 50-60 % of the 100 and 300 nm particles co-localized with the clathrin adaptor AP2 (Figure 4A and B). Interestingly, for beads of 20 and 1000 nm (representing the smallest and the largest


14

Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sizes), few to no beads were found to be associated with the endocytic machinery, respectively (Figure 4A and B). These results indicate that the recruitment of the clathrin machinery to nanoparticles is tightly linked to their size. This is in agreement with previous reports showing that nanoparticles of 100 nm are mostly internalized by CME whereas diameter larger than 500 nm and smaller than 50 nm enter cells through clathrin-independent mechanisms13,14,41. Quantification of the dynamics of CCP association with the 100 and 300 nm beads revealed that AP2 is recurrently recruited. The number of clathrin machinery recruitments on the beads appeared to be dependent on the size of the particles. 300 nm beads displayed a lower number of AP2-GFP recruitments (5.6 ± 1.2 events every 10 min) compared to 100 nm beads (7.5 ± 1.5 events every 10 min) (Figure 4C, S4D and movie 2 and 3). The clathrin machinery was recruited every 117.38 ± 38.46 s for 300 nm beads, every 83.25 ± 29.49 s for 100 nm in comparison to every 74.8 s ± 37.8 s for the virus particles (Figure 5A and 2D). These results demonstrate that the size of immobilized nanoparticles not only dictates the recruitment of the clathrin machinery but also influences the regulatory mechanisms leading to this recruitment, as the number of CME recruitment events is dependent on particle size. The final size of the endocytic structure remains “imprinted” in the clathrin coat at early stage of CCP formation Since the lifetime of a clathrin coat often is associated with its final size,2,31,42, , we next determined if the observed size-dependency for the CCPs lifetime (Figure 4C) was directly correlated with bead size. Detailed analysis revealed that the average time to assemble the endocytic clathrin structure is inversely correlated with bead size. Clathrin coat assembly took on average 59.0 ± 21.5 s on virus particles, 88.5 ± 22.0 s for 100 nm beads and 106.7 ± 25.7 s for 300 nm beads (Figure 5B and 2D). These results strongly suggest that the diameter of the


15


Page 16 of 49

immobilized nanoparticles directly influences the size of the clathrin coated structure growing above them. To further estimate the size of the endocytic structures formed on nanoparticles, we quantified the amount of AP2-GFP recruited on viruses and beads. The fluorescence intensity of a clathrincoated structure is in fact considered directly correlated with the size of a clathrin structure (diameter and relative number of clathrin or adaptor proteins present)3,43. Additionally, we and others, using Correlative Light and Electron Microscopy (CLEM) have recently demonstrated a direct correlation between fluorescence intensity and size of CCSs44,45. Thus, monitoring the maximum fluorescence intensity of CCPs gives a reliable measurement of the final size of the endocytic structures (clathrin/adaptor content). Fluorescence quantification was normalized against CCPs forming within the same cells that did not co-localize neither with beads nor with virus particles (empty pits) (Figure 2B). Analysis of all endocytic events in multiple cells revealed that on average clathrin structures localized on top of the beads were 3.2 ± 1.3 and 5.5 ± 1.1 fold larger for the 100 and 300 nm beads compared to empty pits, respectively (Figure 5C). Similarly, CCPs forming on top of virus particles were almost twice the size of empty pits (1.7 ± 0.7 times greater) (Figure 5C). Of note, we also observed residual clathrin and adaptor proteins (denoted as “MIN” in Figure 5C) after each endocytic event. Interestingly, the amount of these residual clathrin coat proteins was proportional to the size of the nanoparticles. The presence of leftover clathrin molecules at discrete plasma membrane sites after recurrent CCV formation, commonly referred as “hotspots”, has been previously described, but its function remains elusive46,47. Our results suggest that the residual clathrin coat observed during each cycle of coat assembly might act as a nucleator, coordinating spatially and temporally the assembly of a subsequent clathrin structure to the nanoparticles, thereby contributing to creating the observed recurrent recruitment of the clathrin machinery (i.e. formation of a hotspot).


16

Page 17 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The fluorescence analysis of CCP dynamics demonstrates that the size of the clathrin-coated structures directly correlates with the size of viruses and nanoparticles, although these particles are not internalized by the endocytic structures and remain covalently linked to the glass surface. To verify the presence of properly assembled CCPs above the 100 and 300 nm beads, we performed stimulated emission depletion (STED) super-resolution microscopy. As previously reported31,48,49, we observed the presence of ring-like structures, characteristic of clathrin coat, for both the 100 and 300 nm beads (Figure 6). These observations strongly suggest that genuine CCPs are formed on immobilized nanoparticles. This finding corroborates our previous observation describing the recruitment of dynamin at the end of all recurrent endocytic events (Figure S5). Finally, to fully demonstrate the presence of mature CCVs forming above immobilized nanoparticles and to verify our fluorescence quantification results, cells seeded on coverslips coated with clicked beads were examined by transmission electron microscopy (TEM). The embedded cells were sectioned perpendicular to the coverslip in order to visualize the endocytic structures at the basal side of the cells (Figure 7A). In the ultrathin serial sections, single clathrin coated structures were observed; these were always positioned right above the nanoparticles on the cytosolic side of the plasma membrane. Interestingly, for 100 and 300 nm beads, we could identify preserved clathrin coats from the sequential stages of CME, from formation of CCP to pit dissociation (Figure 7B). Importantly, the size of the clathrin-coated structures appeared to be related to the size of the underlying nanoparticles. Although it is important to note that for larger particles (300 nm) the size of the clathrin endocytic coat forming above the nanoparticles appears smaller than the particles themselves. This strongly suggests that the clathrin coat has an intrinsic size limit for the formation of an endocytic structure. Taking together, we propose that the final size of the endocytic structure is “imprinted” in the clathrin coat at early stages of CCP formation. The presence of polystyrene nanoparticles or viral


17


Page 18 of 49

particles of a certain diameter may impose a specific curvature on the cell membrane, which in turn induces the recruitment of the clathrin endocytic machinery. At first, the clathrin coat grows with a curvature radius proportional to the diameter of the particles. The CCP forms and follows the membrane curvature induced by the particles. As the coat grows in size, the CCP is prevented from wrapping around the immobilized nanoparticle, causing the clathrin machinery to disengage, yet grow to a size that is initially imprinted by the cargo at the onset of CCP nucleation.

CONCLUSION In this work we developed a novel approach to investigate early virus-cell interactions. Strategies to immobilize virus particles on surfaces were already described before 20; our method, exploiting the free amine functions at the surface of virus capsids and avoiding genetic modifications, displays a faster experimental set up and can be easily applied to a broader range of viruses. The click-chemistry based-strategy developed in this study gave us the unique chance to dissect chemical and mechanical signaling involved in early clathrin coat recruitment at the single particle level. In this study, we show how immobilized viruses and nanoparticles of a specific size induce a recurrent recruitment of the clathrin endocytic machinery apparently independently from receptor signaling. We propose that the curvature imposed by single viruses and nanostructures can favor CME recruitment. These findings are in agreement with previous studies that show that curvature imposed by external structures, such as nanopillar or nanocones, can induce CME recruitment16,17. Certainly, in the case of viruses, we cannot completely exclude the presence of a receptor-mediated signaling that can further favor the endocytic machinery recruitment and virus internalization. We believe that our method can be applied in future studies


18

Page 19 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aiming at investigating how receptors, together with virus size, can induce the recruitment of the cellular endocytic machinery. Since in our strategy cargo molecules cannot be internalized but can still actively interact with the cell surface, we were able to demonstrate that early events affect later stages of CME. Our results suggest that the initial nanoparticles/virus interaction with the clathrin machinery imprints the initial growth curvature to the coat and determines CCPs maturation and the final size of the CCVs, independently from cargo internalization. Why and how the initial curvature imposed by particles can dictate the final size of CCVs remains unclear. We hypothesize that while in flat clathrin arrays clathrin molecules can be dynamically reorganized 31,45 to drive the transition from flat to curved coat, the presence of an external curvature imposes a “locked” arrangement of clathrin molecules which defines a non-modifiable curvature of the coat. Given the angle existing between each clathrin heavy chains within a clathrin triskelion, the flat clathrin lattice organization might be thermodynamically less stable and can be ultrastructurally rearranged to form curved lattices. On the contrary, in the case of physically induced CCPs, the curvature imposed by the particles for the clathrin lattice to initiate its assembly as a curved, thermodynamically more stable lattice. Here, we propose that immobilized nanoparticles and viruses of size ranging from 80 to 300 nm, induce a specific local membrane curvature resulting in the recruitment and induction of CME. Since the nanoparticles cannot be internalized, the local membrane curvature remains; this could explain the leftover clathrin between each recurrent endocytic cycle and as such promote the recurrent aspect of the observed process. All these aspects could not be investigated with current cell biology techniques, thus emphasizing the innovative aspect of our approach, which we believe can be further applied to the field of nanotechnology, endocytosis and virology.


19


Page 20 of 49

MATERIAL AND METHODS Virus production and purification Reovirus T3D strain was produced by infecting suspension of L-cells with a T3D stock originally obtained from B. N. Fields. Virus particles were pre-purified from L-cells by sonication and freon (1,1,2‐trichloro‐1,2,2‐trifluoroethane; Sigma) extraction; virus particles were then purified through ultracentrifugation on Cesium Chloride (CsCl) gradient and stored in virus buffer (150 mM NaCL, 10 mM MgCl2, and 10 mM Tris‐HCl, ph 7.5) as previously described 50.

Virus and Latex Beads chemical modification 100 μl of reovirus particles (from 1013 particles/ml stock) were mixed with 0.4 μl of Alexa647 NHS Ester (8 mM starting concentration) (Thermo Fisher Scientific) and 0.4 μl of Alkyne-NHS linker (8.3 mM starting concentration) (Iris –Biotech) for 1 h at room temperature (RT). To remove the unbound linker/fluorophores, virus particles were then purified by gel filtration (7K molecular weight cutoff, Invitrogen). Virus particles quantification by light scattering was performed using the Nanosight machine NS300 (Malvern). A total of 1,004 non-modified virus particles and 544 Alexa647/Alkyne-virus particles were counted. Aliphatic Amine Latex Beads, 2% w/v, of 20, 100, 300 and 1000 nm were purchased from Thermo Fisher Scientific. The chemical modification of the beads was performed similarly to virus particles except for the following modifications: For 20 nm beads: final concentrations of 20 mM and 41.5 mM for Alexa647 NHS Ester and Alkyne-NHS linker were used, respectively. For 100 nm beads: final concentrations of 0.08 mM and 20.75 mM for Alexa647 NHS Ester and


20

Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Alkyne-NHS linker were used, respectively. For 300 nm beads: final concentrations of 0.5 mM and 83 mM for Alexa647 NHS Ester and Alkyne-NHS linker were used, respectively. For 1000 nm beads: final concentrations of 0.64 mM and 124.5 mM for Alexa647-NHS Ester and AlkyneNHS linker were used, respectively. For STED imaging, beads were labeled with Atto647 NHS Ester (Sigma) (0.08 mM final concentrations).

Silane-PEG-Azide coating of glass coverslips Pegylation of glass coverslips was performed as previously described24. Briefly, 24 mm glass coverslips (high precision glass coverslips No. 1.5H, Marienfeld Lauda-Königshofen, Germany) were cleaned for 1 h in freshly prepared piranha solution (3:1 H 2SO4/H2O2), washed three times with deionized water, and dried in a stream of nitrogen. Surfaces were then immersed into a 0.125 mM solution of Silane-PEG(3000)-Azide in dry toluene; dry triethylamine was added to a final concentration of 25 M. The reaction mix was then heated at 80°C overnight under nitrogen atmosphere. Finally, glass coverslips were sonicated for 5 min in ethyl acetate and 5 min in methanol, and dried under a nitrogen stream.

Click reaction Click reaction between azide coated glass coverslips and alkyne functions on virus particles was performed as previously described24. Briefly, azide-coated glass coverslips were placed in a freshly prepared reaction mixture consisting of 100 mM L-ascorbic acid, 100 mM Tris HCl (pH 8.5), 1 mM CuSO4 with 1010 Alexa647-virus-Alkyne particles for 1.5 h at RT. Samples were then washed three times with PBS and re-incubated for a second click reaction to covalently bind the cRGDfK peptides (BioTrend) on the glass coverslips. 0.05 mM Alkyne-cRGDfK were clicked


21


Page 22 of 49

for 30 min at RT. Glass coverslips were then washed three times and stored in PBS at 4°C. The same procedure was applied to click latex beads on azide-coated coverslips. Depending on the size, the following number of beads particles are added into the mix: 1012, 1011 1010 and 108 for 20, 100, 300 and 1000 nm beads respectively.

Cell culture and cell lines BSC1 cells (from ATCC), HeLa cells (ATCC) and U373 (kindly provided by Tom Kirchhausen, Harvard Medical School, Boston, USA) were kept in DMEM (Gibco) Media containing 10% fetal bovine serum, 1% vol/vol of penicillin and streptomycin (Gibco) at 37°C and 5% CO2. To obtain U373 cells stably expressing AP2-GFP, cells were transfected with a plasmid encoding the sigma 2 subunit of AP2 fused to GFP and subjected to G418 selection (2 μg/ml) (GIBCO). Genome edited SK-MEL-2 hCLTAen/DNM2en (a kind gift from David G. Drubin, Department of Molecular and Cell Biology, University of California, USA) were maintained in DMEM/F12 (Gibco) Media containing 10% fetal bovine serum, 1% vol/vol of penicillin and streptomycin (Gibco) at 37°C and 5% CO2. Suspensions of L-cells (from ATCC) for virus production were maintained in Joklik MEM medium (Sigma-Aldrich) supplemented with 1% L-Glutamine (Gibco.), 2% FBS (Gibco) 2% Neonatal calf serum (Gibco) and 1% vol/vol of penicillin and streptomycin (Gibco) at 35°C.

Transfection and selection Cell transfection was performed with LipofectamineTM 3000 (Invitrogen) following manufacturer’s protocol. Cells were seeded the day before into a 6 well plate in order to reach the 80-90% of confluence the day of transfection. 2 μg of DNA (for AP2-GFP) or 0.5 μg of DNA (for clathrin light chain-tomato) were mixed in a tube together with 125 μl of Opti-MEM


22

Page 23 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Gibco) and 5 μl of P3000 reagent; 4 μl of Lipofectamine 3000 was diluted into a second tube with 125 μl of Opti-MEM. The two tubes were then mixed together, incubated 10 min at RT and the solution was added onto the cells. Cells were incubated with transfection solution for 8-10 h; the media was then replaced with fresh media. For transient transfection, cells were imaged the day after transfection; for stable cells lines, cells were kept under selection starting from two days after transfection.

Antibodies and plasmids Antibody against reovirus was generated against the reovirus μNS protein by GenScript USA. Vinculin antibody was obtained from Sigma-Aldrich. Secondary antibody Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 568 goat anti mouse were obtained from Molecular probes. JAM-A antibody was obtained from Santa Cruz Biotech (10 μg/ml concentration used for blocking experiments). For testing virus infectivity and replication through LICOR assay we used secondary anti-Rabbit IR800CW (Rockland antibodies) and Draq5 (eBiosciences). To stably or transiently transfect cells, we used mammalian expression vectors fused eGFP to the C-terminus of the rat sigma 2 subunit of AP22 and tdTomato to the N-terminus of the rat clathrin light chain-A10, respectively.

Infectivity studies and indirect immunofluorescence To measure Alexa647/Alkyne virus infectivity we employed the “In-Cell WesternTM” Assay (ICW) from LI-COR technology, which exploits laser-based scanning of near infrared to perform immunofluorescence quantification. Briefly, BSC1 cells were seeded one day prior to the experiment into a 96 well plate in order to reach 80-90% of confluence the day of infection. The day after cells were infected with serial dilutions (from 10 -2 to 10-7) of control virus (virus with


23


Page 24 of 49

no modifications) and Alexa647/Alkyne virus; each dilution is made in triplicate. Negative samples were also included in the assay (cells not infected). 16-18 h post-infection cells were fixed with 2% paraformaldehyde for 15 min at RT. Cells were afterwards permeabilized two times with 0.1% TritonX, and blocked with 1% BSA for 30 min. Samples were then incubated with primary antibodies against reovirus μNS diluted in 1% bovine serum albumin in PBS at RT for 1 h. Coverslips were washed three times with PBS and incubated with the secondary antibody (IR dye 800 anti-mouse for reovirus μNS protein, and Draq5 to stain the DNA) for 1 h at RT. Finally, coverslips were washed three times in PBS. Afterwards the plate was scanned using an Odyssey infrared imaging system and quantitative values from 700 nm and 800 nm excitation channels were exported. First, fluorescence values from 700 nm channel (Draq5) were analyzed to confirm that cell density was the same all over the 96 wells (data not shown). To normalize fluorescence values from 800 nm excitation channel (reovirus μNS protein), first, fluorescence intensity in negative samples was measured; the average value + 3 times the standard deviation was considered as the lowest threshold to determine virus infectivity (0%). The average of intensity values from the highest dilution of cells infected with control virus (virus with no modifications) was considered as 100% infection. All data were then normalized accordingly and plotted as average of three independent experiments. To test Alexa647/Alkyne virus replication efficiency, BSC1 cells were seeded the day before the experiment into a 24 well plate in order to reach 80-90% of confluence the day of infection. The day after, cells were infected with same titer of control virus and Alexa647/Alkyne virus. 3-4 days after infection, the time required by reovirus to replicate and generate newly infectious particles, cells were disrupted by repeated cycles of freezing and thawing. Samples were then


24

Page 25 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


centrifuged (2500 rpm form 10-15 min) and the supernatant containing newly produced virus particles was used to infect BSC1 cells previously seeded into a 96 well plate. To test infectivity we performed an ICW assay from LI-COR technology as described above. Results were plotted as average of three independent experiments. For indirect immunofluorescence assay, BSC1 cells were seeded the day before the experiment onto a 12 mm diameter glass coverslips (Marienfeld, Lauda-Königshofen, Germany) in order to reach 70-80% of confluence the day of infection. Virus solutions were then added onto the cells; cells were incubated for 16-18 h and then fixed with 2% paraformaldehyde for 15 min at RT. To test Alexa647/ alkyne virus infectivity in the presence of copper, the virus solution was incubated 1 h at RT with 1 mM CuSO4; afterwards the virus was purified by gel filtration (7K molecular weight cutoff, Invitrogen) and added to cell cultures. Fixed samples were washed three times in PBS and permeabilized with 0.05% Triton-X100 for 15 min at RT. Cells were blocked with 1% bovine serum albumin in PBS for 30 min at RT. Samples were then incubated with primary antibodies diluted in 1% bovine serum albumin in PBS at RT for 1 h. Coverslips were washed three times with PBS and incubated with the secondary antibody for 1 h at RT. Finally, coverslips were washed three times in PBS, rinsed in water and mounted in ProLong Gold Antifade mounting medium supplemented with 4,6‐diamidino‐2‐phenylindole (Molecular Probes). Slides were then imaged by epifluorescence using a Nikon Eclipse Ti-S (Nikon) microscope (20X and 40 magnification). For JAM-A experiments, HeLa cells were seeded onto a 12 mm diameter glass coverslips one day before the experiment in order to reach 70-80% of confluence the day of infection. Cells were then incubated 1 h at RT with 10 μg/ml JAM-A antibody solution in PBS. Cells were then washed and incubated 30 min on ice with virus solutions in PBS. Finally, cells were washed


25


Page 26 of 49

again in PBS and incubated 16-18 h in normal growth medium before being fixed in 2% PFA. Indirect immunofluorescence assay was performed as described above.

Live-cell microscopy To perform live-cell microscopy we used a spinning disc confocal microscope, as it was previously shown to perfectly suit live imaging of AP2 expressing cell lines, offering an optimal signal-to-noise ratio essential for dynamics and quantification analysis 2,10,11,51,52,53. Cells were seeded on 24 mm diameter coverslips coated with clicked virus or beads and cRGDfK and livecell microscopy was performed 6 h post-seeding. Live-cell imaging was performed with an inverted spinning-disk confocal microscope (PerkinElmer) using oil immersion objectives (60x, 1.42 numerical aperture, Apo TIRF, Nikon or 100x, 1.4 numerical aperture, Plan Apo VC, Nikon) and a CMOS camera (Hamamatsu Orca Flash 4). Cells, objectives and microscope stage were kept at 37°C and 5% CO2 through the presence of an environment-control chamber. Cells were imaged for 5 or 10 min with a frame interval of 3 sec/frame. To perform live-cell imaging of HeLa cells in the presence of JAM-A antibody, a suspension of Hela cells was first incubated 1 h with anti-JAM-A antibody (final concentration 10 μg/ml); afterwards, cells were seeded onto a virus coated coverslip previously glued to the bottom of a multi-well chamber. In this way, control cells and cells treated with anti-JAM-A antibody were seeded onto the same virus coated coverslip and imaged in parallel. Cells were imaged 6 h post-seeding; JAM-A antibody was added into the sample every 1.5 h (final concentration 10 μg/ml). After imaging, the same concentration of virus was added into cells treated with anti-JAM-A antibody and control cells. The cells were incubated 30 min on ice with virus solutions in PBS. Finally, cells were washed again in PBS and incubated 16-18 h in


26

Page 27 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


normal growth medium before being immunostained for reovirus infectivity (protocol as described before).

SEM Sample preparation and imaging Coverslips coated with clicked viruses were fixed in 2% glutaraldehyde in PBS for 15 min at RT; afterwards critical point drying of virus particles was performed using a CPD 030 Critical point dryer (Bal-Tec). Samples were finally sputter-coated with a titanium-gold layer using a Leica ACE600 machine. Coverslips presenting clicked beads were fixed in 2% paraformaldehyde for 15 min at RT; samples were directly sputter-coated with a carbon layer using a Leica EM ACE200. Samples were imaged by using a ZEISS SEM Leo1530.

TEM sample preparation and imaging Cells growing on coverslips coated with 100 and 300 nm beads were fixed in 2.5% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 supplimented with 2% sucrose, 50 mM KCl, 2.6 mM MgCl2, 2.6 mM CaCl2, for 30 min at RT and at 4˚C overnight. After rinsing in buffer the samples were further fixed in 1% osmium in cacodylate buffer, washed in water, and incubated in 0.5% uranylacetate in water for 30 min. Dehydration was performed in 10 min steps in an ethanol gradient followed by Spurr resin embedding and polymerization at 60˚C. The blocks were cut in 70 nm thin serial-sections using a Leica UC6 ultramicrotome (Leica Microsystems Vienna) and collected on pioloform coated slot grids. The post-stained sections were imaged on a JEOL JEM-1400 electron microscope (JEOL, Tokyo) operating at 80 kV and equipped with a 4K TemCam F416 (Tietz Video and Image Processing Systems GmBH, Gautig).


27


Page 28 of 49

STED sample preparation and imaging U373 AP2-GFP cells were seeded on coverslips presenting 100 or 300 nm clicked-beads (labeled with Atto647N). Cells were fixed 6 h post seeding and mounted using a ProLong Gold Antifade mounting medium (Molecular Probes). STED imaging was performed using the Leica TCS SP8 STED 3X system (Leica Microsystems GmbH, Mannheim, Germany) with a 100x, 1.4 numerical aperture STED White objective. Gated STED signal of AP2-GFP was generated with 488 nm excitation from a white light laser (WLL) and 592 nm depletion. Atto647N was excited with 633 nm from the WLL and depleted with 775 nm pulsed laser.

Image analysis and quantification In order to count the number of virus particles on modified/not modified glass surfaces and to measure virus fluorescence distribution, the pixel classification and object classification workflow in Ilastik (http://ilastik.org) was used. To measure fluorescence intensity distribution of Alexa647/Alkyne –virus clicked or deposited on glass, a total of 62,204 Alexa647/Alkyne virus deposited on glass and 12,965 Alexa647/Alkyne-virus clicked on azide coated coverslips were counted and mean intensity were measured. Fluorescence intensity values were normalized to the average intensity from each group (Alexa647-virus and) and plotted in bins of 0.5 relative fluorescence unit; the percentage of total particles falling within each bin is shown on the y-axis. Same procedure was adopted to measure fluorescence intensity distribution of Alexa647-virus and Alexa647/Alkyne-virus deposited on glass. A total of 11,001 Alexa647-virus and 35,027 Alexa647/Alkyne-virus deposited on glass were counted and mean intensity were measured; data are normalized and plotted as described above. In order to measure the size of virus particles and polystyrene beads from the micrographs obtained with SEM, the pixel classification and object classification workflow in Ilastik


28

Page 29 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(http://ilastik.org) was used. A total of 655 of non-modified virus particles and 629 Alexa647/Alkyne-virus particles were counted. CME events were tracked using Fiji (https://fiji.sc/). For the analysis of AP2-GFP signal on virus/beads spots, spots of interest were manually listed into a ROI manager and fluorescence intensity over time was automatically measured. To track AP2-GFP signal over time from empty pits (pits not co-localizing with virus/beads spots) the TrackMate plugin from Fiji was used. Data were normalized to the average highest fluorescence intensity of empty pits in each cell. After normalization, quantification analysis was performed. We extracted for each virus/beads track, the value with highest fluorescence intensity (MAX-fluo) and the value with the lowest intensity (MIN-fluo). The abovementioned measurements were calculated for three cells per condition (virus, 100 and 300 nm beads).

Fitting of Gaussian profiles into fluorescence signal captured by live-cell microscopy The normalized AP2-GFP fluorescence signal from virus/beads spots, empty pits and random spots served as input to the interactive peak fitter (IPF version 8.4) program in Matlab (MathWorks,

USA)

under

command

line

(https://terpconnect.umd.edu/~toh/spectrum/InteractivePeakFitter.htm).

mode Random

(peakfit.m)54 spots

were

collected by unsupervised selection of pixels within each cell imaged with live-cell microscopy. The number of empty pits and random spots was set to be ~3 times higher than the virus/beads spots for further analysis. Because each fluorescence signal from virus/bead spots was composed to up to 12 maxima within the time window of acquisition, 1-13 Gaussian functions were fitted to each track. Gaussian functions with fixed width significantly outperformed other functions (e.g. Lorenzian; Gaussian with variable width) in fitting as reported by goodness-of-fit measures (r2, percentage error of fit of the fitted curve composed of Gaussian functions) (data not shown).


29


Page 30 of 49

Therefore, the fluorescence signal was deconvolved with Gaussian models of fixed width. The exact number of Gaussian functions explaining each profile was chosen based on goodness-of-fit measures (Figure S3). To avoid overfitting of the Gaussian functions, visual inspection of the fits was performed to finally select the number of Gaussians (number of events). Because Gaussian profiles fitted also to the background signal, a cut-off (average of MIN-fluo of virus/beads per cell + stand deviation) of fluorescence intensity was chosen to remove those events. Then, we defined three measures for modeling the fluorescence signal: (a) Number of recruitment events of clathrin machinery that is described by counting the number of Gaussian profiles fitted per virus/bead track and statistically compared to empty and random spots. (b) Rate of peak occurrence (time between two cycles of AP2 GFP recruitment) that is described by the time window in sec between two concurrent maxima of Gaussian functions fitted to the fluorescence signal. (c) Lifetime of pits (time of a complete cycle of AP2 GFP recruitment) that is described by the fixed width of the Gaussian functions fitted per track. The abovementioned measures were calculated per track and their distributions are then plotted in a comparative manner.

ACKNOWLEDGEMENTS This work was supported by the German Science Foundation (SFB1129 projects P15 to E.A.C.A and J.P.S, and P14 to S.B.). The support of the Max Planck Society is greatly acknowledged. S.B. is supported by the Chica and Heinz Schaller foundation. We thank Dr. Panagiotis Kastritis (EMBL, Heidelberg) for providing essential support for image analysis, Dr. Anna Luise Grab (Heidelberg University, Institute of Physical Chemistry) for support during SEM imaging and Dr. Vibor Laketa, (Heidelberg University Clinic, Department of Infectious Diseases) for support with spinning disc microscopy. We acknowledge the EM Core Facility at Heidelberg University for assistance with Electron microscopy.


30

Page 31 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR CONTRIBUTIONS S.B., E.A.C.A. and J.P.S conceived the project and discussed the results. S.B., E.A.C.A. and M.F. designed and discussed the experiments. M.F. performed the experiments, analyzed the data and made the figures. M.F. performed SEM imaging. T.W. participated to the establishment of the reovirus click reaction protocol. C.F. performed TEM imaging. Z.J. performed STED imaging. P.N.M.S. performed virus dynamic light scattering analysis. S.B., E.A.C.A. and M.F. wrote the manuscript. All authors read and approved the final manuscript.

SUPPORTING INFORMATION AVAILABLE Supplemental Figures S1-6 and Movies 1-3 are available free of charge via the Internet at http`://pubs.acs.org

ABBREVIATION CME, clathrin-mediated endocytosis; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; PM, plasma membrane; CuAAC, copper (I)-catalyzed alkyne-azide cycloaddition; MOI, multiplicity of infection; μNS, Reovirus non-structural protein; Si-PEG-N3, silane-PEG(3000)Azide; cRGDfK, cyclo[Arg-Gly-Asp-D-Phe-Lys]; GFP, green fluorescent protein; JAM-A, junctional adhesion molecule A; TEM, transmission electron microscopy; STED, stimulated emission depletion.

AUTHOR INFORMATION


31


Corresponding

authors:

[email protected]

Page 32 of 49

or

[email protected]

REFERENCES (1)

Mcmahon, H. T.; Boucrot, E. Molecular Mechanism and Physiological Functions of Clathrin-Mediated Endocytosis. Nat. Publ. Gr. 2011, 12, 517–533. doi:10.1038/nrm3151.

(2)

Ehrlich, M.; Boll, W.; Van Oijen, A.; Hariharan, R.; Chandran, K.; Nibert, M. L.; Kirchhausen, T. Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits. Cell 2004, 118, 591–605. doi:10.1016/j.cell.2004.08.017.

(3)

Cocucci, E.; Aguet, F.; Boulant, S.; Kirchhausen, T. The First Five Seconds in the Life of a Clathrin-Coated Pit. Cell 2012, 150, 495–507. doi:10.1016/j.cell.2012.05.047.

(4)

Kadlecova, Z.; Spielman, S. J.; Loerke, D.; Mohanakrishnan, A.; Reed, D. K.; Schmid, S. L. Regulation of Clathrin-Mediated Endocytosis by Hierarchical Allosteric Activation of AP2. 2016. doi:10.1083/jcb.201608071.

(5)

Taylor, M. J.; Perrais, D.; Merrifield, C. J. A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis. PLoS Biol. 2011, 9. doi:10.1371/journal.pbio.1000604.

(6)

Ferguson, S. M.; De Camilli, P. Dynamin, a Membrane-Remodelling GTPase. Nat. Rev. Mol. Cell Biol. 2012, 13, 75–88. doi:10.1038/nrm3266.

(7)

Godlee, C.; Kaksonen, M. From Uncertain Beginnings: Initiation Mechanisms of ClathrinMediated Endocytosis. J. Cell Biol. 2013, 203, 717–725. doi:10.1083/jcb.201307100.

(8)

Kaksonen, M.; Roux, A. Mechanisms of Clathrin-Mediated Endocytosis. Nat. Rev. Mol. Cell Biol. 2018. doi:10.1038/nrm.2017.132.

(9)

Grove, J.; Marsh, M. The Cell Biology of Receptor-Mediated Virus Entry. J. Cell Biol. 2011, 195, 1071–1082. doi:10.1083/jcb.201108131.

(10)

Cureton, D. K.; Massol, R. H.; Saffarian, S.; Kirchhausen, T. L.; Whelan, S. P. J. Vesicular Stomatitis Virus Enters Cells through Vesicles Incompletely Coated with Clathrin That Depend upon Actin for Internalization. PLoS Pathog. 2009, 5. doi:10.1371/journal.ppat.1000394.

(11)

Cureton, D. K.; Massol, R. H.; Whelan, S. P. J.; Kirchhausen, T. The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly during Clathrin-Dependent Endocytosis. PLoS Pathog. 2010, 6. doi:10.1371/journal.ppat.1001127.

(12)

Boulant, S.; Stanifer, M.; Lozach, P.-Y. Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis. Viruses 2015, 7, 2794–2815. doi:10.3390/v7062747.


32

Page 33 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13)

Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Size-Dependent Internalization of Particles via the Pathways of Clathrin- and Caveolae-Mediated Endocytosis. Biochem. J. 2004, 377, 159–169. doi:10.1042/BJ20031253.

(14)

Pricop, D.; Andrieş, M. Endocytosis and Exocytosis of Gold Nanoparticles. Int. J. Nano 2014, 25, 1–9. doi:10.2147/IJN.S26592

(15)

Boulant, S.; Stanifer, M.; Kural, C.; Cureton, D. K.; Massol, R.; Nibert, M. L.; Kirchhausen, T. Similar Uptake but Different Trafficking and Escape Routes of Reovirus Virions and Infectious Subvirion Particles Imaged in Polarized Madin-Darby Canine Kidney Cells. Mol Biol Cell 2013, 24, 1196–1207. doi:10.1091/mbc.E12-12-0852.

(16)

Galic, M.; Jeong, S.; Tsai, F.-C.; Joubert, L.-M.; Wu, Y. I.; Hahn, K. M.; Cui, Y.; Meyer, T. External Push and Internal Pull Forces Recruit Curvature-Sensing N-BAR Domain Proteins to the Plasma Membrane. Nat. Cell Biol. 2012, 14, 874–881. doi:10.1038/ncb2533.

(17)

Zhao, W.; Hanson, L.; Lou, H.-Y.; Akamatsu, M.; Chowdary, P. D.; Santoro, F.; Marks, J. R.; Grassart, A.; Drubin, D. G.; Cui, Y.; Cui, B. Nanoscale Manipulation of Membrane Curvature for Probing Endocytosis in Live Cells. Nat. Nanotechnol. 2017, 12, 750–756. doi:10.1038/nnano.2017.98.

(18)

Soto, C. M.; Blum, A. S.; Vora, G. J.; Lebedev, N.; Meador, C. E.; Won, A. P.; Chatterji, A.; Johnson, J. E.; Ratna, B. R. Fluorescent Signal Amplification of Carbocyanine Dyes Using Engineered Viral Nanoparticles. J. Am. Chem. Soc. 2006, 128, 5184–5189. doi:10.1021/ja058574x.

(19)

Sakin, V.; Paci, G.; Lemke, E. A.; Müller, B. Labeling of Virus Components for Advanced, Quantitative Imaging Analyses. FEBS Lett. 2016, 590, 1896–1914. doi:10.1002/1873-3468.12131.

(20)

Kwak, E.-A.; Jaworski, J. Controlled Surface Immobilization of Viruses via Site-Specific Enzymatic Modification. J. Mater. Chem. B 2013, 1, 3486. doi:10.1039/c3tb20526f.

(21)

Wang, Q.; Li, K.; Chen, Y.; Li, S.; Nguyen, H. G.; Niu, Z.; You, S.; Mello, C. M.; Lu, X. Chemical Modification of M13 Bacteriophage and Its Application in Cancer Cell Imaging. Bioconjug. Chem. 2010, 21, 1369–1377. doi:10.1021/bc900405q.

(22)

Shah, P. N. M.; Stanifer, M. L.; Höhn, K.; Engel, U.; Haselmann, U.; Bartenschlager, R.; Kräusslich, H.-G.; Krijnse-Locker, J.; Boulant, S. Genome Packaging of Reovirus Is Mediated by the Scaffolding Property of the Microtubule Network. Cell. Microbiol. 2017, e12765. doi:10.1111/cmi.12765.

(23)

Ivanovic, T.; Boulant, S.; Ehrlich, M.; Demidenko, A. A.; Arnold, M. M.; Kirchhausen, T.; Nibert, M. L. Recruitment of Cellular Clathrin to Viral Factories and Disruption of Clathrin-Dependent Trafficking. Traffic 2011, 12, 1179–1195. doi:10.1111/j.16000854.2011.01233.x.

(24)

Schenk, F. C.; Boehm, H.; Spatz, J. P.; Wegner, S. V. Dual-Functionalized Nanostructured Biointerfaces by Click Chemistry. Langmuir 2014, 30, 6897–6905.


33


Page 34 of 49

doi:10.1021/la500766t. (25)

Ruoslahti, E. RGD AND OTHER RECOGNITION SEQUENCES FOR INTEGRINS. Annu. Rev. Cell Dev. Biol. 1996, 12, 697–715. doi:10.1146/annurev.cellbio.12.1.697.

(26)

Wilcox, M. E.; Yang, W.; Senger, D.; Rewcastle, N. B.; Morris, D. G.; Brasher, P. M.; Shi, Z. Q.; Johnston, R. N.; Nishikawa, S.; Lee, P. W.; Forsyth, P.A. Reovirus as an Oncolytic Agent against Experimental Human Malignant Gliomas. J. Natl. Cancer Inst. 2001, 93, 903–912.

(27)

Massol, R. H.; Boll, W.; Griffin, A. M.; Kirchhausen, T. A Burst of Auxilin Recruitment Determines the Onset of Clathrin-Coated Vesicle Uncoating. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 10265–10270. doi:10.1073/pnas.0603369103.

(28)

Virshup, D. M.; Bennett, V. Clathrin-Coated Vesicle Assembly Polypeptides: Physical Properties and Reconstitution Studies with Brain Membranes. J. Cell Biol. 1988, 106, 39 LP-50.

(29)

Schulz, W. L.; Haj, A. K.; Schiff, L. A. Reovirus Uses Multiple Endocytic Pathways for Cell Entry. 2012, 86, 12665–12675. doi:10.1128/JVI.01861-12.

(30)

Kural, C.; Kirchhausen, T. Live-Cell Imaging of Clathrin Coats. Methods in enzymology, 2012, 505, 59–80. doi:10.1016/B978-0-12-388448-0.00012-7.

(31)

Bucher, D.; Frey, F.; Sochacki, K. A.; Kummer, S.; Bergeest, J.-P.; Godinez, W. J.; Krausslich, H.-G.; Rohr, K.; Taraska, J. W.; Schwarz, U. S.; Boulant, S. Clathrin-Adaptor Ratio and Membrane Tension Regulate the Flat-to-Curved Transition of the Clathrin Coat during Endocytosis. Nat. Commun. 2018, 9, 1109. doi: 10.1038/s41467-018-03533-0.

(32)

Boulant, S.; Kural, C.; Zeeh, J.-C.; Ubelmann, F.; Kirchhausen, T. Actin Dynamics Counteract Membrane Tension during Clathrin-Mediated Endocytosis. Nat. Cell Biol. 2011, 13, 1124–1131. doi:10.1038/ncb2307.

(33)

Loerke, D.; Mettlen, M.; Yarar, D.; Jaqaman, K.; Jaqaman, H.; Danuser, G.; Schmid, S. L. Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation. PLoS Biol. 2009, 7. doi:10.1371/journal.pbio.1000057.

(34)

Liu, A. P.; Aguet, F.; Danuser, G.; Schmid, S. L. Local Clustering of Transferrin Receptors Promotes Clathrin-Coated Pit Initiation. J. Cell Biol. 2010, 191, 1381–1393. doi:10.1083/jcb.201008117.

(35)

Barton, E. S.; Forrest, J. C.; Connolly, J. L.; Chappell, J. D.; Liu, Y.; Schnell, F. J.; Nusrat, A.; Parkos, C. A.; Dermody, T. S. Junction Adhesion Molecule Is a Receptor for Reovirus. Cell 2001, 104, 441–451. doi:10.1016/S0092-8674(01)00231-8.

(36)

Maginnis, M. S.; Forrest, J. C.; Kopecky-Bromberg, S. a; Dickeson, S. K.; Santoro, S. a; Zutter, M. M.; Nemerow, G. R.; Bergelson, J. M.; Dermody, T. S. Beta1 Integrin Mediates Internalization of Mammalian Reovirus. J. Virol. 2006, 80, 2760–2770. doi:10.1128/JVI.80.6.2760-2770.2006.


34

Page 35 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37)

Maginnis, M. S.; Mainou, B. A.; Derdowski, A.; Johnson, E. M.; Zent, R.; Dermody, T. S. NPXY Motifs in the 1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry. J. Virol. 2008, 82, 3181–3191. doi:10.1128/JVI.01612-07.

(38)

Janssen, W. E.; Rios, A. M. Non-Specific Cell Binding Characteristics of Para-Magnetic Polystyrene Microspheres Used for Antibody-Mediated Cell Selection. J. Immunol. Methods 1989, 121, 289–294. doi:10.1016/0022-1759(89)90173-7.

(39)

Chithrani, B. D.; Chan, W. C. W. Elucidating the Mechanism of Cellular Uptake and Removal of Protein-Coated Gold Nanoparticles of Different Sizes and Shapes. Nano Lett. 2007, 7, 1542–1550. doi:10.1021/nl070363y.

(40)

Mironava, T.; Hadjiargyrou, M.; Simon, M.; Jurukovski, V.; Rafailovich, M. H. Gold Nanoparticles Cellular Toxicity and Recovery: Effect of Size, Concentration and Exposure Time. Nanotoxicology 2010, 4, 120–137. doi:10.3109/17435390903471463.

(41)

Shang, L.; Nienhaus, K.; Nienhaus, G. U. Engineered Nanoparticles Interacting with Cells: Size Matters. J. Nanobiotechnology 2014, 12, 5. doi:10.1186/1477-3155-12-5.

(42)

Kirchhausen, T. Imaging Endocytic Clathrin Structures in Living Cells. Trends Cell Biol. 2009, 19, 596–605. doi:10.1016/j.tcb.2009.09.002.

(43)

Saffarian, S.; Cocucci, E.; Kirchhausen, T. Distinct Dynamics of Endocytic ClathrinCoated Pits and Coated Plaques. PLoS Biol. 2009, 7. doi:10.1371/journal.pbio.1000191.

(44)

Avinoam, O.; Schorb, M.; Beese, C. J.; Briggs, J. A. G.; Kaksonen, M. Endocytic Sites Mature by Continuous Bending and Remodeling of the Clathrin Coat. Science 2015, 348, 1369–1372. doi:10.1126/science.aaa9555.

(45)

Lampe, M.; Vassilopoulos, S.; Merrifield, C. Clathrin Coated Pits , Plaques and Adhesion. J. Struct. Biol. 2016, 196, 48–56. doi:10.1016/j.jsb.2016.07.009.

(46)

Lampe, M.; Pierre, F.; Al-Sabah, S.; Krasel, C.; Merrifield, C. J. Dual Single-Scission Event Analysis of Constitutive Transferrin Receptor (TfR) Endocytosis and LigandTriggered 2-Adrenergic Receptor ( 2AR) or Mu-Opioid Receptor (MOR) Endocytosis. Mol. Biol. Cell 2014, 25, 3070–3080. doi:10.1091/mbc.E14-06-1112.

(47)

Bottanelli, F.; Kromann, E. B.; Allgeyer, E. S.; Erdmann, R. S.; Wood Baguley, S.; Sirinakis, G.; Schepartz, A.; Baddeley, D.; Toomre, D. K.; Rothman, J. E.; Bewersdorf, J. Two-Colour Live-Cell Nanoscale Imaging of Intracellular Targets. Nat. Commun. 2016, 7, 10778. doi:10.1038/ncomms10778.

(48)

Bates, M.; Huang, B.; Dempsey, G. T.; Zhuang, X. S-Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science (80-. ). 2007, 317, 1749– 1753. doi:10.1126/science.1146598.

(49)

Furlong, D. B.; Nibert, M. L.; Fields, B. N. Sigma 1 Protein of Mammalian Reoviruses Extends from the Surfaces of Viral Particles. J. Virol. 1988, 62, 246–256.

(50)

Boucrot, E.; Saffarian, S.; Zhang, R.; Kirchhausen, T. Roles of AP-2 in Clathrin-Mediated


35


Page 36 of 49

Endocytosis. 2010, 5. doi:10.1371/journal.pone.0010597. (51)

Ferguson, J. P.; Willy, N. M.; Heidotting, S. P.; Huber, S. D.; Webber, M. J.; Kural, C. Deciphering Dynamics of Clathrin-Mediated Endocytosis in a Living Organism. J. Cell Biol. 2016, 214, 347–358. doi:10.1083/jcb.201604128.

(52)

Wood, L. A.; Larocque, G.; Clarke, N. I.; Sarkar, S.; Royle, S. J. New Tools for “HotWiring” Clathrin-Mediated Endocytosis with Temporal and Spatial Precision. J. Cell Biol. 2017, 1–15. doi:10.1083/jcb.201702188.

(53)

O’Haver, T. A Pragmatic Introduction to Signal Processing; 2014.


36

Page 37 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC figure.


37


Page 38 of 49

Figure 1. Covalent immobilization of viral particles on glass surfaces. A) Schematic of reovirus immobilization on glass surfaces. 1) Reovirus particles were fluorescently labeled with Alexa647 dye and coupled to an alkyne linker. Through CuAAC (Click chemistry), virus particles were covalently bound onto Si-PEG-N3 coated coverslips. 2) To allow for proper cell adhesion on the surfaces, cyclic-RGDfK-alkyne peptides were clicked onto the same surface. B) Alexa647-virus or Alexa647/Alkyne-virus were clicked on Si-PEG-N3 coated glass coverslips in


38

Page 39 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence or absence of copper (Cu). The conjugation efficiency was estimated using fluorescence microscopy by counting the number of immobilized virus particles/area (3.9 x 10 4 µm2). C) Alexa647/Alkyne-viruses were either deposited on glass or clicked on Si-PEG-N3 coated coverslips. The fluorescence intensities of the labeled viral particles were measured and plotted as described in methods. Distribution of normalized fluorescence mean intensity (in arbitrary units) of particles was quantified for non-clicked (n=62204) and clicked particles (n=12965). D) BSC-1 cell were infected with CuSO4 pre-incubated Alexa647/Alkyne-virus at MOI 1 (sample 1), or seeded on clicked-virus coated coverslips (sample 2), or were incubated with no virus (sample 3). 16-18 hours post-infection, cells were fixed and stained for reovirus infection. The ratio of infected cells to total number of cells was normalized to cells infected with Alexa647/Alkyne-virus pre-incubated with CuSO4. Data are shown as mean value ± standard deviation (SD) of three independent replicates.


39


Page 40 of 49

Figure 2. The endocytic clathrin machinery is recruited on clicked viruses. A) Fluorescence live-cell imaging of U373 cells expressing AP2-GFP (green) seeded on clicked Alexa647/ Alkyne virus (red) coated coverslips. Insert region 1 is a representative example of virus particles


40

Page 41 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


co-localizing with the clathrin machinery; insert region 2 corresponds to a CCP without a virus particle. The number of viral particles co-localizing with the clathrin machinery was normalized to the total number of viruses located under each cell (mean and SD from three cells are shown). B) Kinetic intensity profiles and schematic representations (in the above inserts) of a representative CCPs (green) colocalizing with a single immobilized viral particle (red) (upper panel) and of a CCP not containing a virus particle (empty pit) (lower panel). Virus intensity profiles exhibit several “peaks” which represent recurrent recruitments of the clathrin machinery above the immobilized virus particles. The AP2-GFP fluorescence intensity for each time point was normalized to the average of the maximum AP2-GFP fluorescence intensity of empty pits in the same cell (see method section for details). C) Normalized number of AP2 recruitment events on virus particles (87 spots), Random spots (271 spots) and empty pits (271 spots). The number of AP2 recruitment events from each intensity profile (collected from virus spots, empty pits and random spots) was measured and plotted. Virus spots displayed on average a higher number of AP2 recruitment events compared to empty pits and random spots. Average of recruitment events: Empty pits, 2.3 +/- 2.3; random spots, 4.9 +/- 4.0; virus spots, 8.1 +/- 2.0. The x-axis shows the number of CCP recruitments over 10 min. Data collected from three cells. D) (Upper panel) Normalized distribution of the rate of peak occurrence onto clicked viruses. For every intensity profile collected from virus spots, the time distance between each peak of AP2 was measured and plotted (illustration in the insert; black arrows represent the time distance between each peak of AP2 within one intensity profile). Data generated from 87 virus particles. (Lower panel) Normalized distribution of the lifetimes of CCPs onto clicked viruses. For every intensity profile collected from virus spots, the lifetime of each peak of AP2 was measured and plotted (illustration in the insert; black arrows represent the lifetime of every peak of AP2 within one intensity profile). Data generated from 87 virus particles.


41


Page 42 of 49

Figure 3. CCP recruitment on virus particles is JAM-A independent. A) Hela cells were preincubated with PBS (control) or PBS + JAM-A antibody (10 g/ml) and afterwards infected with reovirus at MOI=1 (see method section for details); virus infection was monitored by indirect immunofluorescence using an anti-μNS antibody (green). Infectivity was measured by


42

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


counting the percentage of infected cells; data were normalized to cells infected by reovirus in absence of JAM-A neutralizing antibody. Data are shown as mean value ± SD. B) Hela cells expressing CLC-tomato (in green) were preincubated for 1 h at RT with PBS or PBS + JAM-A. Cells were seeded on clicked Alexa647/Alkyne virus (in red) coated coverslips (see method section for details) and live-cell imaging was performed. C) (Upper panel) The number of viral particles co-localizing with the clathrin machinery was normalized to the total number of viruses located under each cell. Data are shown as mean value ± SD from five cells per conditions. p value < 0.05 was considered significant (unpaired t-test). (Lower panel) Relative number of infected cells in control and in samples treated with JAM-A neutralizing antibody after infection post-live imaging (see method section for details). D) Kinetic intensity profiles of a representative CCP (green) co-localizing with a single immobilized viral particle (in red) in control cells (upper panel) and in cells treated with JAM-A neutralizing antibody (lower panel) (frame rate of data acquisition of 3 sec for 10 min). E) Normalized frequency of clathrin recruitment to virus particles in control cells (black columns) (95 virus spots) and in cells treated with JAM-A neutralizing antibody (grey columns) (87 virus spots). Five cells per condition were analyzed.


43


Page 44 of 49

Figure 4. Recruitment of CCP to nanoparticles depends on their sizes. A) Fluorescence livecell imaging of U373 cells expressing AP2-GFP (green) seeded on 20, 100, 300 and 1000 nm clicked beads (red). Inserts are representative examples of beads and AP2 in each sample; (lower part) kinetic intensity profiles of representative CCPs co-localizing with single immobilized beads. The AP2-GFP fluorescence intensity for each time point was normalized to the average of the maximum AP2-GFP fluorescence intensity of empty pits in the same cell (see method section for details). B) Percentage of beads co-localizing with AP2-GFP. The number of beads colocalizing with the clathrin machinery was normalized to the total number of beads located under each cell (mean and SD from four cells are shown; p value < 0.05 is considered significant, unpaired t-test). C) Normalized frequency of AP2 recruitment on 100 and 300 nm beads (n=99


44

Page 45 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and n=105 respectively) and empty pits (n=333 and n=329 respectively). The x-axis shows the number of AP2 recruitment per 10 min. Data collected from three cells per condition.


45



Page 46 of 49

46

Page 47 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Dynamics and quantification of AP2 recurrent recruitment on beads and viruses. A) Normalized distribution of the rate of peak occurrence (time distance between peaks of AP2; illustration in the insert). B) Normalized distribution of coated pits lifetime (time duration of each peak, illustration in the insert). Data were generated from 87 virus particles, 99 and 105 beads of 100 and 300 nm, respectively. C) Fluorescence intensity quantification from live-cell imaging of U373 expressing AP2-GFP (green) seeded on clicked-viruses or clicked-beads. The AP2-GFP fluorescence intensity for each time point was normalized to the average of the maximum AP2-GFP fluorescence intensity of empty pits into the same cell (see method section for details). The Max fluorescence intensity from viruses and beads represents the average of the maximum values of AP2 recruited on each particle. The Min fluorescence intensity represents the residual amount of AP2 left behind after completion of each AP2 recurrent recruitment (illustration in inset). Data are shown as the mean value ± SD. p value < 0.05 is considered significant (unpaired t-test).


47


Page 48 of 49

Figure 6. STED imaging of U373 AP2-GFP cells seeded on clicked beads. Cells were seeded on coverslips coated with 100 nm (upper part) and 300 nm (lower part) clicked-beads and fixed with paraformaldehyde 6 h post-seeding. Samples were then mounted and imaged using STED microscopy. Image analysis reveals the presence of AP2 “ring structures” around clicked beads, Green: AP2. Red: Alexa647/Alkyne clicked beads.

Figure 7. The size of nanoparticles imprints the final size of CCVs A) TEM images of CCPs from ultra-thin sections of U373 cells seeded on glass coverslips with no beads. Cells were seeded upon glass coverslips; 6 h post-seeding, cells were fixed and processed for TEM imaging (see method section for details) (scale bar 100 nm). B) TEM images from ultra-thin sections of


48

Page 49 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


U373 cells seeded upon bead coated coverslips (100 nm beads (upper), 300 nm beads (lower). Cells were seeded upon clicked-beads coated coverslips; 6 h post-seeding cells were fixed and processed for TEM imaging (see method section for details). Sequence of pictures shows different stages of clathrin coated pit assembly upon immobilized beads (scale bar 100 nm). 1) Clicked beads 2) CCP growing on top of beads.


49

Surface Immobilization of Viruses and Nanoparticles Elucidates Early

Recommend Documents