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Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking En-Ze Sun, An-An Liu, Zhi-Ling Zhang, Shu-Lin Liu, Zhi-Quan Tian, and Dai-Wen Pang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b07853 • Publication Date (Web): 29 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking

En-Ze Sun, An-An Liu, Zhi-Ling Zhang, Shu-Lin Liu, Zhi-Quan Tian, Dai-Wen Pang*

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, P.R. China

*Corresponding author Email: [email protected]

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Abstract Entry is the first critical step for the infection of influenza A virus and of great significance for the research and development of anti-flu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of single-virus tracking technique and quantum dots, we have realized real-time and multi-color visualization of the endocytic process of individual viruses and comprehensive dissection of two distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of virus, with a large population of events for short-lived recruitments of these two proteins being abortive. In addition, the differentiated durations of dynamin recruitment and responses to inhibitors in these two routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also

participates

in

the

maturation

of

clathrin-coated

pit

in

the

clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the 2


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mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.

Keywords: influenza A virus, quantum dot, tracking, endocytosis, dynamin, clathrin

Influenza A virus (IAV) causes periodical epidemics and pandemics responsible for considerable casualties every year, and it becomes one of the most hazardous global public health threats.1 Due to its great medical significance, IAV has been studied as a paradigm to investigate the mechanism governing the animal virus infection and to promote the development of anti-virus strategies for several decades. As its first step in infection, IAV attaches to host cell by its major envelope protein, hemagglutinin (HA), which binds to terminal sialic acid residue exposed on the surface of cell.1 Afterwards, IAV exploits host cell’s endocytosis to pass through the plasma membrane (PM) and is delivered from the periphery to the perinuclear region of cell. The virus eventually releases its genome into cytoplasm by fusing with the membrane of late endosome.2 The first defined entry route of IAV is clathrin-mediated endocytosis (CME), which is also responsible for uptake of many protein cargoes, such as transferrin, epidermal growth factor and low-density lipoprotein,3 and is common to be 3


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hijacked by several pathogens for their entry.2,4 In addition to CME, a few studies also reported the existence of clathrin-independent endocytic route on different experimental conditions, such as macropinocytosis, in the past decade.5-9 During endocytosis, membrane fission is essential for the transition of a membrane pit into an endocytic vesicle. Since dynamin, a large multidomain GTPase, promotes the membrane fission in CME and some other types of endocytoses,10 it is probably functioning during clathrin-dependent entry of IAV. But whether dynamin plays any role in the clathrin-independent entry of IAV is still controversial and needs further investigation.5-7 Most of previous studies on virus infection, which relied on ensemble and fixed cell assays,5,11,12 could only provide global information about the average state of the whole population of viruses and reflect discontinuous moments in the complicated infection process. Given the dynamic and stepwise properties of the entry process of IAV, conventional methods are difficult to address some obscure but critical issues, such as the preference in different entry routes, the recruitment and function of involved proteins during IAV entry and the adaptation of endocytic machinery during the uptake of IAV, an unconventional cargo. The fluorescence microscopy-based single-virus tracking (SVT) technique offers the means to answer above questions. Since it’s capable to simultaneously monitor the motility of individual viruses and the consecutive recruitment of proteins to cellular structures, SVT technique would be 4


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well-suited for revealing the transient but indispensable events during virus entry and displaying a continuous and precise image of the interaction between virus and cellular components.13-17 Furthermore, statistical analysis of the diversity in the entry behaviors of multiple viruses could provide us with previously unavailable information about the underlying mechanism.9,17-19 Similar approaches have already been employed to improve the precision in the characterization of the endocytosis of protein cargoes, especially for the machinery of CME.20-24 The prerequisite of tracking a single virus in a living cell is labeling the viruses with fluorescent materials. Semiconductor quantum dots (QDs), due to their outstanding brightness, superior resilience to photobleaching and tunable emission wavelengths, are a kind of ideal labeling material for real-time and multi-color bioimaging.25,26 In our previous works, we established a series of facile strategies to synthesize quantum dots of different compositions27-31 and label viral envelope with QDs efficiently,32,33 which enabled us to simultaneously track multiple viruses in one cell, giving rise to maximized performance of SVT approach.34,35 So far, this QD-based SVT technique has already been used in real-time visualization and revealing mechanisms for the infection of multiple species of viruses.36,37 The goal of this study was to dissect distinct endocytic routes of IAV by analyzing the dynamics of its entry processes. We monitored the QDs-labeled viruses and fluorescent protein (FP)-labeled cellular endocytic structures in 5


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Madin-Darby canine kidney (MDCK) cells at a time resolution of 1 s by a spinning-disk confocal microscope system. Compared with the total internal reflection fluorescence (TIRF) microscope which is generally used to observe the events on the basal plasma membrane, spinning-disk confocal microscope is more suitable for SVT technique, because virus with diameter of a hundred or more nanometers is difficult to get access to the ventral surface of adherent cell, while spinning-disk confocal microscope can be used to observe the entire process of virus infection, from the apical surface to the perinuclear region of a cell. Based on comprehensive and quantitative analyses of the entry behaviors of IAV in both clathrin-dependent and clathrin-independent routes, we determined the preference in these two routes and the dependency of dynamin during virus entry, and established multistep models for both entry routes. Moreover, we discovered a large subpopulation of events for IAV-induced recruitments of clathrin and dynamin being short-lived and abortive, and further proposed the functional difference of dynamin in these two entry routes.

Results Visualization of Single IAV and Endocytic Structure on Plasma Membrane IAV (strain A/chicken/Hubei/01-MA01/1999(H9N2)) which propagated in embryonated eggs was purified by ultracentrifugation. The intactness of IAV was retained completely with representative pleiomorphy in the electron 6


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micrograph (Figure 1A),38 indicating that the harvested viruses could be used in subsequent SVT experiments. The viral envelopes were biotinylated with biotinylation reagent and then conjugated with streptavidin-QDs (SA-QDs) through the reaction of biotin and streptavidin as described.32 Neither the biotinylation nor the conjugation with SA-QDs did significantly impair the infectivity of IAV (Figure 1B). The labeling efficiency of SA-QDs was further determined by the colocalization assay of the fluorescence signals of antibody against HA protein and SA-QDs conjugated to virus. Over 90% of the viruses were labeled with SA-QDs, whether the viruses were fixed on the glass-bottomed Petri Dish (93.2 ± 2.5%, Figure 1C) or attached to the PM of MDCK cells (94.1 ± 3.5%, Figure 1D).

Figure 1. Visualization of single IAV and endocytic structure. (A) Electron micrograph of purified IAV. Scale bar, 200 nm. (B) Average titers of wildtype, biotinylated and QDs-labeled IAV, respectively. Error bars represent SD of three independent experiments. (C and D) Representative IF images of QDs-labeled (605 nm) and wildtype viruses fixed on the glass-bottomed Petri Dish (C) and attached on the PM of a MDCK cell (D), respectively. (E) Representative IF images of CCSs on the PMs of MDCK cells expressing AcGFP1-Clc (Anti-Clc, AcGFP1 and Merge) and endogenous CCSs on the PMs of normal MDCK cells (Anti-Clc-normal). Scale bars, 10 μm.

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In addition to the virus, the cellular endocytic structures were also fluorescently labeled. A fusion protein of clathrin light chain a and AcGFP1 (AcGFP1-Clc) was expressed in MDCK cells to visualize the clathrin-coated structures (CCSs) on the PM which were generated by recruiting clathrin from cytoplasm. In the confocal images, the fluorescence signals of AcGFP1-Clc were represented as scattered puncta as the signals of antibody against clathrin (Figure 1E), demonstrating that AcGFP1-Clc was successfully incorporated to CCSs. Their colocalization efficiency was 93.7 ± 2.1%. The distributions of both the AcGFP1-labeled CCSs in the transfected cells and the endogenous CCSs in normal cells were uniform throughout the PM (Figure 1E), and the expression of AcGFP1-Clc didn’t impact the uptake of transferrin, a typical CME-specific cargo (Figure S1A), indicating that the function of Clc was not affected by the fusion with AcGFP1 and that expressing AcGFP1-Clc in MDCK cells was suitable for monitoring the formation and degradation of CCSs in the cell.

The Predominant ClathrinClathrin-Dependent Entry Route of IAV In order to identify where the entry of IAV took place in confocal image, CellMask PM stain, an impermeable amphipathic dye was employed. We focused our analyses on the viruses whose diffusions were integrally within the focal plane. The colocalization of QDs-labeled viruses with dye puncta in the cytoplasm indicated that viruses were internalized along with the endocytic 8


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vesicles derived from PM (Figure S2A). Their movements were coincident throughout the internalization process (Figure S2B-D and Video S1), indicating that IAV hijacked the cellular endocytosis for its infection. Dual-color observation was subsequently performed to visualize the entry of QDs-labeled IAV in the MDCK cells expressing AcGFP1-Clc (Figure 2A and Videos S2, S3). The trajectory of virus represented the motion of virus from the PM to the perinuclear region of cell (Figure 2D). The velocity of the virus was aligned with the fluorescence intensity of the corresponding AcGFP1-labeled CCS, which represented the time course of clathrin recruitment, to delineate a whole picture of the entry process of a virus (Figures 2C and S3A, B). As an indication of the initiation of clathrin-coated pits (CCP), the clathrin recruitment occurred at the binding sites of viruses. The fluorescence intensity of clathrin increased gradually, nearly plateaued, and then peaked, indicating the generation and gradual maturation of a CCP. The subsequent drastic decline and eventual disappearance of the fluorescence signal of clathrin were followed by the onset of viral rapid movement toward the cytoplasm, suggesting the successful endocytosis of the virus into a clathrin-coated vesicle (CCV) and the rapid uncoating of CCV before the intercellular trafficking of the virus-containing vesicle. In addition, the calculated mean square displacement (MSD) of the virus demonstrated that it mainly experienced a slow (ca. 0.1 μm/s) and restricted diffusion during the clathrin recruitment (Figure 2E). The virus was confined to the binding site when the 9


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CCP was generating and growing. The rapid movement (> 0.5 μm/s) of virus conformed with the property of motor-driven directed diffusion, representing the cytoskeleton-dependent transportation of virus-containing vesicle (Figure 2F).32 These two stages of diffusion were also observed during the internalization of virus in the PM-stained cells (Figure S2E-H). Statistical results showed that ca. 85% (137/161 in 20 cells) of the internalized viruses were endocytosed with a period of clathrin recruitment (Figure 2B), indicating that IAV preferred to trigger and hijack CME for its entry.

Figure 2. Clathrin-dependent entry of IAV. (A) Snapshots of the entry of a QDs-labeled (605 nm) virus via CME shown in Video S2 (highlighted in white squares). Scale bar, 2 μm. (B) Proportion of viruses that recruit clathrin during entry in the group of internalized viruses (n=161). (C) Fluorescence intensity curve of clathrin at the site of virus and velocity curve of viral diffusion shown in A. (D) Trajectory of viral diffusion shown in A. The restricted and directed phases are shown in red and blue, respectively. Scale bar, 1 μm. (E and F) Dependence of MSD against time during viral diffusion. The red (E) and blue (F) lines represent the fits to corresponding equations of restricted and directed diffusion, respectively.

According to the fluorescence intensity of clathrin as well as the diffusion mode of virus, we divided the time course of clathrin-dependent IAV entry into 10


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three phases (Figure S4I): an “assembly phase” corresponding to the increase of fluorescence intensity, as clathrin accumulated to assemble lattices caging the growing membrane pits; a “disassembly phase” corresponding to the decrease of fluorescence intensity, as clathrin lattices disassembled from nascent vesicles; followed by a “pre-directed phase” corresponding to the time gap between the end of clathrin recruitment and the onset of viral directed diffusion. Since the rapid and directed diffusion mainly represented the cytoskeleton-dependent transportation of virus-containing vesicles, the pre-directed phase implied the recruitment and modulation of the cytoskeleton for the internalization of nascent vesicles.39

Asynchronous Recruitments of Clathrin and Dynamin during IAV Entry Dynamin plays critical roles in several types of endocytoses including CME.10 It could assemble into helix structure winding around the neck of membrane pit to “pinch off” the pit into an endocytic vesicle by the energy from GTP hydrolysis. As dynamin-2 (Dyn2) was the only isoform ubiquitously expressed in mammalian cells,10 we expressed Dyn2-AcGFP1 in MDCK cells, which didn’t affect the uptake of transferrin (Figure S1B). We monitored the entry of QDs-labeled viruses in MDCK cells co-expressing mCherry-Clc and Dyn2-AcGFP1 to investigate the interaction between dynamin and virus during clathrin-dependent IAV entry (Figure 3A, B and Videos S4, S5). Consistent with aforementioned results, ca. 87% (118/135 in 20 cells) of the internalized 11


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viruses entered the cells with the assistance of CME (Figure 3H). As for dynamin, this proportion was 88% (119/135 in 20 cells), indicating that besides clathrin, IAV entry was also predominantly dynamin-dependent. This conclusion was further strengthened by the observation of IAV entry in cells solely expressing Dyn2-AcGFP1 (Figure S4A-E and Videos S6, S7), which obtained close proportion of dynamin-dependent entry events (ca. 92%, 163/178 in 20 cells, Figure S4F-H). These reproducible results not only proved the robust dependence of clathrin and dynamin during IAV entry, but also demonstrated the negligible influences of different FPs, QDs and excitation lasers.

Figure 3. Asynchronous recruitments of clathrin and dynamin during IAV entry. (A) Snapshots of the entry of a QDs-labeled (705 nm) virus via clathrin- and dynamin-dependent endocytosis shown in Video S4 (highlighted in the white squares). Scale bar, 5 μm. (B) Kymograph of the binding site of the virus shown in A. The t1-t5 segments stand for the five phases which are also represented in corresponding colors in C, D and G. (C) Fluorescence intensity curves of clathrin and dynamin at the site of virus and velocity curve of viral diffusion shown in A. (D) Trajectory of viral diffusion shown in A. The restricted and directed phases are shown in red and blue, respectively. Scale bar, 2 μm. Inset: Enlarged trajectories of five phases during the restricted diffusion of virus. Scale bar, 0.5 μm. (E and F) Dependence of MSD against time during 12


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viral diffusion. The red (E) and blue (F) lines represent the fits to corresponding equations of restricted and directed diffusion, respectively. (G) Distribution of duration in each phase. Error bars represent SD among trajectories (n=69). Boxes represent the data from the 25th to 75th percentile. (H) Proportion of viruses that recruit clathrin and/or dynamin during entry in the group of internalized viruses (n=135).

The

nearly

complete

coincidence

of

clathrin-dependent

and

dynamin-dependent entry events (116 events, Fig 3H) indicated the indispensable role of dynamin in the clathrin-dependent entry route of IAV. We subsequently examined the temporal order and the duration of their recruitments in this continuous process. To this end, we complemented the time course of dynamin recruitment into above three phases of clathrin recruitment, generating a five-phase model for the clathrin-dependent entry of IAV as follows (Figure 3B-D), which demonstrated the asynchronization in the recruitments of clathrin and dynamin during IAV-hijacked CME. 1) “Initiation phase” (t1) corresponded to the initiation of clathrin recruitment and CCP assembly prior to dynamin recruitment. Dynamin wasn’t involved in this early stage of CCP formation, which also mainly caused the assembly phase of dynamin recruitment was shorter than that of clathrin recruitment (Figure S4I). 2) “Assembly phase of dynamin” (t2) corresponded to the accumulation of 13


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dynamin till peak while the fluorescence intensity of clathrin nearly plateaued or kept increasing. The fluorescence intensity of dynamin kept in a relatively low level initially, and then burst sharply, indicating that the nascent CCP was fully mature and recruited dynamin to pinch off itself into a CCV. 3) “Disassembly phase of dynamin” (t3) was characterized as the rapid descending of the fluorescence intensity of dynamin after peak. Generally, the fluorescence intensity of clathrin also started to decline in this phase, indicating that both the dynamin helix and the clathrin lattices began to disassemble successively after the transition of CCP to CCV. 4) “Continued disassembly phase of clathrin coat” (t4) corresponded to the drop of fluorescence intensity of clathrin after the end of dynamin recruitment indicating the ongoing uncoating process of CCV. The signal of dynamin generally disappeared prior to that of clathrin, but in a few cases, clathrin uncoating completed ahead (Figure 3F). The recruitments of both clathrin and dynamin occurred during the restricted stage of virus movement (Figure 3C-E). 5) “Pre-directed phase” (t5) was as same as the last phase in the three-phase model of clathrin recruitment discussed above. The directed diffusion of virus might initiate prior to the disappearance of clathrin’s signal sporadically (Figure 3F and G).

IAVAV-Induced Abortive Recruitments of Clathrin and Dynamin During the observation of clathrin-dependent entry of IAV, we found that not 14


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every virus which induced clathrin recruitment could be finally internalized. A large subpopulation of viruses stayed in the restricted diffusion phase after the end of clathrin recruitment, instead of triggering directed diffusion (Figure 4A and Video S8). Remarkably, in these cases, the pattern of clathrin recruitment resembled that in the productive CCPs which successfully transformed into virus-containing CCVs (Figure 4B and C), but the duration of clathrin recruitment was about one third shorter (Figure 4G). These results suggested that the clathrin coats were still assembled at the beginning, but these growing CCPs failed in the transition into CCVs and degraded eventually. Thereby the viruses in these abortive CCPs remained on the PM. We further examined the number of virus-induced abortive CCPs, which was 17.1 per cell, as twice as the productive ones (Figure 4H). Therefore, the majority of virus-induced CCPs were abortive. Interestingly, as for dynamin, such abortive recruitment with shorter duration was prevailing as well (Figure 4D-H and Video S9). Therefore, the decline of fluorescence signal of dynamin represented the departure of dynamin from degraded membrane pits instead of nascent virus-containing vesicles.

Figure 4. Abortive recruitments of clathrin and dynamin induced by IAV. (A and D) Snapshots of abortive recruitments of clathrin (A) and dynamin (D) at the sites of QDs-labeled (605 nm) viruses shown in Videos S8 and S9, respectively (highlighted in white squares). Scale bars, 2 μm. (B and E) 15


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Trajectories of corresponding viral diffusions shown in A and D, respectively. Scale bars, 0.05 μm. Insets: Dependence of MSD against time during viral diffusion. The red lines represent the fits to equation of restricted diffusion. (C and F) Fluorescence intensity curves of clathrin (C) and dynamin (F) at the sites of viruses and velocity curves of viral diffusions shown in A and D, respectively. (G) Average duration of abortive and productive recruitment events of clathrin and dynamin, respectively. Error bars represent SD among trajectories (n=171, 80, 156 and 89). (H) Distribution of the numbers of abortive and productive recruitment events of clathrin and dynamin per cell, respectively. Error bars represent SD among cells (n=10 and 10). Boxes represent the data from the 25th to 75th percentile.

Given the strong correlation between the recruitments of clathrin and dynamin in IAV entry events and the prevalence of IAV-induced abortive recruitments of both proteins, their abortive recruitments in triple-color observation were further analyzed (Figure 5A, B and Video S10). As in productive CCPs, their recruitments in abortive CCPs were asynchronous as well (Figure 5C). In agreement with the results of dual-color observation, events of virus-induced abortive recruitment outnumbered those of productive recruitment (Figure 5D). The predominance of clathrin- and dynamin-positive portion in the abortive events demonstrated the strong correlation between the abortive recruitments of clathrin and dynamin. 16


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Figure 5. Asynchronization and correlation of the abortive recruitments of clathrin and dynamin. (A) Snapshots of the asynchronous and abortive recruitments of clathrin and dynamin at the site of a QDs-labeled (705 nm) virus shown in Video S10 (highlighted in white squares). Scale bar, 2 μm. (B) Trajectory of restricted diffusion of virus shown in A. Scale bar, 0.2 μm. Inset: Dependence of MSD against time during viral diffusion. The red line represents the fit to equation of restricted diffusion. (C) Fluorescence intensity curves of clathrin and dynamin at the site of virus and velocity curve of viral diffusion shown in A. (D) Distribution of the number of events of abortive and productive recruitments per cell, respectively. Error bars represent SD among cells (n=10). Boxes represent the data from the 25th to 75th percentile.

Recruitment Recruitment of Dynamin during ClathrinClathrin-Independent Entry of IAV We further investigated the possibility of IAV entry in cells whose CME was inhibited (CME-negative cell). Overexpressing the clathrin binding domain (carboxyl terminus, residues 380-696) of AP180 protein (AP180-C), an accessory protein in CME, could exert a dominant-negative effect on CME function through trapping clathrin.40 The uptake of transferrin was blocked severely in the cells overexpressing AP180-C (Figure S5), indicating that cellular CME function was effectively inhibited. Unlike transferrin, IAV could still be internalized in the cells overexpressing 17


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mCherry-AP180-C and co-expressing Dyn2-AcGFP1, indicating that there was indeed a clathrin-independent entry route of IAV in MDCK cells (Figure 6A). Notably, some of the viruses were colocalized with dynamin puncta on the PM. The SVT image demonstrated that the internalized virus recruited dynamin for its entry (Figures 6B, S6 and Videos S11 and S12). Statistical results showed that ca. 92% (35/38 in 12 cells) of the internalized viruses recruited dynamin for their entry (Figure 6C), indicating that this clathrin-independent entry route of IAV was dynamin-dependent, too. Compared with the case in cells solely expressing Dyn2-AcGFP1, the number of virus entry events in the CME-negative cells reduced about two thirds (Figure 6D), suggesting the relatively low entry efficiency of IAV in the clathrin-independent route, which might in turn contribute to the preference of clathrin-dependent entry route during IAV infection.

Figure 6. Dynamin-dependent entry

of IAV via clathrin-independent

endocytosis. (A) Representative fluorescence images of the QDs-labeled (705 nm) virus internalized in the CME-negative cells co-expressing Dyn2-AcGFP1. Scale bar, 10 μm. (B) Snapshots of the entry of a QDs-labeled virus shown in Video S11 that recruits dynamin in the CME-negative cells (highlighted in white squares). Scale bar, 2 μm. (C) Proportion of viruses that recruit dynamin during entry in the group of internalized viruses in CME-negative cells (n=38). (D) Distribution of the number of dynamin-dependent entry events per cell in 18


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CME-positive and CME-negative cells, respectively. Error bars represent SD among cells (n=20 and 12). Boxes represent the data from the 25th to 75th percentile. (E) Fluorescence intensity curve of dynamin at the site of virus and velocity curve of viral diffusion shown in B. (F) Average duration of each phase of virus-induced dynamin recruitment in CME-positive and CME-negative cells, respectively. Error bars represent SD among trajectories (n=89 and 30).

The patterns of dynamin recruitment and viral diffusion are analogous to those in the clathrin-dependent route (Figures 6E and S6E, F). We divided the whole time course of virus entry into three phases as above analysis (Figure 6F). In this case, the duration of assembly phase of dynamin was nearly a half of that in the clathrin-dependent route. The significant difference of the duration of dynamin assembly phase implied the role of dynamin in these two entry routes might be different. In order to substantiate this hypothesis, two inhibitors that aimed at different domains of dynamin and functioned at the different stages of membrane remodeling were introduced into the SVT experiments. Dynasore targets the G domain and interferes its GTPase activity, resulting in that the dynamin helix can’t obtain sufficient energy to drive membrane fission.41 Whereas OcTMAB targets the pleckstrin homology (PH) domain and blocks its interaction with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) on the PM which is crucial for the polymerization of dynamin into helix structure.42 19


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In the cells co-expressing mCherry-Clc and Dyn2-AcGFP1 (CME-positive cells), both Dynasore and OcTMAB treatments made the viruses only attach on the PM without internalization (Figure 7A). Interestingly, several viruses were still colocalized with clathrin and dynamin puncta, suggesting the virus-induced recruitments of clathrin and dynamin. Because of the reversible inhibition effect of both Dynasore and OcTMAB, the virus infection recovered when the inhibitors were removed (Figure S7). Similarly, in the CME-negative cells, Dynasore and OcTMAB treatments also effectively inhibited the internalization of viruses (Figure 7B), which strengthened our SVT results that both entry routes were dynamin-dependent. However, discrete colocalization of viruses with dynamin puncta was hardly observed upon the inhibition of OcTMAB, while Dynasore treatment did not interfere their colocalization (Figure 7B), suggesting that dynamin recruitment was different in these two entry routes of IAV, which were congruent with the much shorter duration of dynamin assembly phase in clathrin-independent route.

Figure 7. Effect of dynamin inhibitors on IAV entry. Representative live-cell fluorescence images of the QDs-labeled (705 nm) virus internalized in the CME-positive (A) and CME-negative cells (B) subjected to 80 μM Dynasore and 30 μM OcTMAB exposure, respectively. White arrow heads mark the sites of colocalization. The regions in white squares are magnified on right. Scale bars, 10 μm. 20


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Discussion Two Distinct DynaminDynamin-Dependent Entry Routes of IAV In the present study, two distinct entry routes of IAV were dissected (Figure 8). The first entry route was clathrin- and dynamin-dependent. Nearly 90% of the viruses utilized this pathway for their entry, which was close to the labeling efficiency of CCS on PM and higher than previous study on the infection process of X31 (H3N2) IAV in BSC-1 cells,9 suggesting that the predominance of IAV entry pathway might depend on cell lines and IAV subtypes. This entry route was also exploited by some other viruses in different manners.14,17,36,37 All the events of virus-induced clathrin recruitment detected in our observation initiated at the binding sites of viruses. Such a de novo manner of CCP initiation was also reported in the entry of vesicular stomatitis virus, a bullet-shaped virus with similar size.17 On the contrary, virus with much smaller size, such as dengue virus, was reported to mainly rely on being captured by pre-existing CCP for its entry.14 These diverse manners of CCP initiation might be attributed to the viral size, since generally bigger virus could cluster more receptors which would enhance CCP nucleation.43

Figure 8. Multistep models of two entry routes of IAV. IAV entry in MDCK cell is predominantly clathrin-dependent. CCP is initiated at the binding site of virus and grows with expansion of clathrin coat. Dynamin is recruited to the 21


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maturating CCP and a selection process takes place subsequently, in which a majority of the CCPs fail and degrade eventually. Dynamin is successively recruited to the neck of productive CCP which passed through the checkpoint to form a helix, which severs the CCP into a virus-containing CCV. The dynamin helix and clathrin coat are rapidly disassembled subsequently. IAV can also enter the cell in a clathrin-independent manner when cellular CME is inhibited. In this case, dynamin is required during the membrane fission as well, but isn’t involved during the growing of membrane pit.

Once the cellular CME was inhibited, IAV switched to a less effective, clathrin-independent but dynamin-dependent entry route with a shorter duration of dynamin recruitment, which was in line with previous studies on the entry of filamentous IAV through Dynasore-sensitive macropinocytosis.7 However, there was also evidence of the serum-induced dynamin-independent macropinocytosis-like endocytosis of IAV.6 The relatively lower entry efficiency of this route seemed no impairment to the productivity of progeny viruses.8 Since the uncertainties in both macropinocytosis itself and its involvement in virus entry,44 the clathrin-independent entry route of IAV deserves further study.

An Adapted ClathrinClathrin-Mediated Endocytosis Hijacked by IAV IAV-hijacked CME and common CME responsible for the uptake of protein 22


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cargoes have similarity in terms of the asynchronous recruitments of clathrin and dynamin,20-22 indicating that the CME process is precisely regulated under a universal machinery. However, the IAV-hijacked CME has some adaptations, demonstrating great flexibility in this machinery. The virus-induced recruitment of clathrin produced “terminal” productive CCP with longer duration, which disappeared once the virus-containing CCV was generated. In contrast, the “non-terminal” CCP, which persisted on the PM after a CCV was generated and was able to continuously recruit clathrin to produce new CCVs at the same site, was usually observed in the common CME.20-22 These disparities were largely attributed to the relatively bulky size of IAV. Typically, the virus strain we used is pleiomorphic with a diameter of about 120 nm, larger than a common CCV in mammalian cells.3 IAV inevitably requires much larger membrane pit to encapsulate it into a vesicle and severely escalates the complexity of the modulation of endocytosis, including the necessity of actin polymerization to provide extra force for membrane fission.39 The non-terminal CCP suits for generating a smaller CCV that would only consume a part of the CCP, thus the rest on the PM could prepare for the next round of endocytosis. As for IAV, an ultra-large object, its endocytosis would exhaust the whole CCP. Therefore, the non-terminal CCP was hardly observed in IAV-hijacked CME.

A Selection Process during ClathrinClathrin-Dependent Entry of IAV According to our observation, only one third of the viruses which induced the 23


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clathrin recruitment could be internalized. The better part of virus-induced CCPs failed before the transition into CCVs and became abortive with shorter duration of recruitment, which could only be detected by SVT technique. In the previous study on the common CME using TIRF microscope, a prevailing subgroup of CCPs with a shorter lifetime was ever observed and assumed to be abortive due to the limitation of TIRF microscopy in the observation of intracellular events.23 Our approach based on spinning-disk confocal microscope enables us to track the generation and motion of a virus-containing CCV both on the PM and in the cytoplasm, and thereby we could distinguish productive and abortive CCPs according to the viral motility, providing a direct evidence of that these short-lived CCPs degraded eventually. Our findings support the proposal that CCP must pass through one or more checkpoints prior to the membrane fission and only a minority of the growing CCPs could survive in this selection and proceed toward CCV.23,24 We have also evidenced the significant correlation and asynchronization between the abortive recruitments of clathrin and dynamin, indicating the participation of dynamin during the maturation of CCP.

The Functional Difference of Dynamin in Two Entry Routes Although dynamin is involved in both entry routes, its function in these two routes may not be entirely same. On one hand, the duration of dynamin assembly phase in clathrin-dependent entry route was nearly twice as long as 24


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that in clathrin-independent entry route. On the other hand, in response to the inhibitor treatments, these two entry routes made a difference in dynamin recruitment. Upon the Dynasore treatment, dynamin recruitment still kept occurring in both CME-positive and CME-negative cells, indicating that dynamin played the membrane-remodeling role in both entry routes. As for the OcTMAB treatment, dynamin recruitment was retained in the CME-positive cells but disturbed in the CME-negative cells, indicating that dynamin was recruited to the site of virus before the CCP was fully mature, which was also in agreement with previous proposal that PH domain mutant of dynamin could be recruited in common CME.45 Moreover, the dynamin recruitment in the abortive and immature CCP also provides a direct evidence of the involvement of dynamin in the maturation of CCP. Several lines of previous evidences also pointed to the additional function of dynamin in regulating the maturation of CCP prior to the membrane fission, basing on the interaction of dynamin with other accessory proteins in CME.10,22,23 In contrast, abortive recruitment of dynamin was hardly observed in the clathrin-independent entry route, suggesting that dynamin might only prompt the membrane fission in this case.

Conclusion In the present study, we accomplished multi-color and real-time visualization of the entry process of IAV in MDCK cells by a combination of efficient virus-labeling method and SVT technique. Thanks to the outstanding 25


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fluorescence properties of QDs, we realized comprehensive and quantitative analyses of the entry behaviors of viruses in two distinct routes. This QD-based SVT technique enables us to establish multistep models for both routes, identify a major abortive subpopulation of IAV-induced CCPs and further propose the functional difference of dynamin in these two routes.

Materials and Methods Cell Line and Virus MDCK ( ATCC® CCL-34) cells were cultured in minimum essential medium (MEM, Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies) and maintained at 37℃ with 5% CO2. Cells were discarded once passaged for several times and new aliquots of frozen cells were thawed. Influenza A virus (strain A/chicken/Hubei/01-MA01/1999(H9N2)) was amplified as previously described.46 In brief, virus-containing allantoic fluid was diluted in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) which was supplemented with 100 μg/mL streptomycin sulfate and 100

U/mL

penicillin

G,

and

then

inoculated

into

10-day-old

specific-pathogen-free embryonated eggs (Merial China Ltd.) which were subsequently incubated at 37℃ for 48 h. The progeny viruses were collected from the allantoic fluid by ultracentrifugation at 110000 × g for 90 min in a Beckman Type70 rotor. The supernatants were then subjected to a 15%-60% 26


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(w/v) discontinuous sucrose gradient and centrifuged at 110000 × g for 1 h in a Beckman SW28 rotor. The resultant viral bands were harvested and resuspended in PBS buffer. The viruses were aliquoted and stored at -80℃ after sucrose was removed from the virus suspension. The titer of purified virus was determined by TCID50 assay with MDCK cells and hemagglutination assay with fresh chicken red blood cells (gift from Dr. Jianjun Chen).46 Purified viruses were dropped on Formvar grids, negatively stained with 2% phosphotungstic acid and subsequently examined on a Hitachi 7000 electron microscope.

Labeling of Influenza A Virus According to previously described approach,32 aliquoted viruses (about 3000 TCID50) were incubated with 0.5 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS buffer at room temperature for 1 h to generate biotinylated viruses. Unreacted biotinylation reagent was removed with a NAP-5 desalting column (GE Healthcare) and then virus aggregates were removed with a 0.22 μ m pore size filter (Millipore) before experiments. Prepared biotinylated viruses bound to MDCK cells seeded in glass-bottomed Petri Dish (Beijing Derunfeng) at 4℃ for preventing internalization. Unbound viruses were washed out with prechilled Tyrode’s plus buffer (135 mM NaCl, 10 mM KCl, 0.4 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5.6 mM glucose, and 0.1% BSA, pH 7.4). Afterward, 2 nM SA-QDs (Wuhan Jiayuan) with emission 27


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maxima of 605 nm or 705 nm (depending on the experimental requirements) were added to the cells at 4℃ to conjugate the biotinylated viruses, and unconjugated QDs were washed out with prechilled Tyrode’s plus buffer.

Plasmid Construction and Transfection The coding sequences of CLTA (Gene ID: 100856559), DNM2 (Gene ID: 484949) and SNAP91 (Gene ID: 403452) genes, which encoded Clc, Dyn2 and AP180, respectively, were amplified from cDNA library of MDCK cells and then cloned into appropriate vectors (pAcGFP1-C1/N1 or pmCherry-C1, Clontech Laboratories) to produce AcGFP1/mCherry-Clc, Dyn2-AcGFP1 and AcGFP1/mCherry-AP180-C (the carboxyl

terminus, residues

380-696)

constructs. MDCK cells were transfected with appropriate amount of indicated plasmid by using Lipofectamine LTX reagent (Life Technologies) according to the manufacturer’s instructions. Transfected cells were seeded into a 35 mm glass-bottomed Petri Dish at approximate 24 h prior to the observation.

Dyes Staining and Inhibitors Treatment CellMask Deep Red Plasma Membrane Stain (Life Technologies), whose fluorescence excitation/emission maxima were 649/666 nm, was used to label the PM of MDCK cell. According to manufacturer’s instructions, the provided 1000 × concentrated stain solution was diluted with Tyrode’s plus buffer to 28


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prepare 0.5 × working solution. MDCK cells in glass-bottomed Petri Dishes were stained with this working solution at 4℃ for 5 min. Afterward, cells were rinsed with prechilled Tyrode’s plus buffer for subsequent virus attachment. Dynasore and OcTMAB (abcam) were dissolved in DMSO and then diluted with Tyrode’s plus buffer to prepare working solutions with final concentrations of 80 μM and 30 μM (with 0.5% DMSO), respectively. Prior to the virus attachment, cells were pretreated with inhibitor at 37℃ for 30 min, and the corresponding inhibitor was maintained throughout the virus labeling and observation until removal. After labeling of virus with SA-QDs, the cells were incubated at 37℃ for 20 min for virus internalization and observed for the first time. Afterward, the inhibitor was washed out, then cells were incubated at 37℃ for another 20 min and observed again.

Transferrin Uptake Assay Tetramethylrhodamine (TMR)-conjugated transferrin (Life Technologies), whose fluorescence excitation/emission maxima were 555/580 nm, was used to determine the function of CME in MDCK cells. Cells were treated with 25 μg/mL TMR-conjugated transferrin at 4℃ for 10 min, shifted to 37℃ for 20 min for internalization if necessary, and fixed to prepare specimens for imaging after washing out the unbound transferrin.

Immunofluorescence and Antibodies 29


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Antibodies used in this study were: mouse monoclonal anti-clathrin antibody (abcam), anti-HA antibody (GeneTex) and Dylight 649 conjugated polyclonal goat-anti-mouse IgG (Abbkine). Cells or viruses were seeded in a glass-bottomed Petri Dish, fixed with 4% (w/v) paraformaldehyde for 20 min, permeabilized in PBS buffer containing 0.1% (v/v) Triton X-100 for 5 min and blocked in PBS buffer supplemented with 10% (v/v) FBS and 3% (w/v) BSA for 30 min. Primary and secondary antibodies were diluted in PBS buffer containing 1% BSA. Cells were incubated with primary antibody at 37℃ for 90 min followed by Dylight 649 conjugated secondary antibody at 37℃ for 45 min. All the specimens were examined on a spinning-disk confocal microscope system and the colocalization efficiency of fluorescence signals from different channels was analyzed with Image J software (NIH Image).

Live-cell Fluorescence Imaging Cells grew on a glass-bottomed Petri Dish and QDs-labeled viruses attached on the cell surface. The medium was replaced with Tyrode’s plus buffer and the Petri Dish was mounted onto a temperature controlled chamber (INUBG2, TOKAI HIT) on the microscope stage. Time-lapse live-cell images were acquired using a spinning-disk confocal microscope system (The Revolution XD, Andor) equipped with a motorized invert microscope (Olympus IX 81), a Nipkow disk type confocal unit (CSU 22, Yokogawa), an Emission 30


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filter wheel (Sutter Instruments), a heated 100 × objective (NA=1.40) and an EMCCD (Andor iXon Ultra 897). DPSS lasers at 488 nm, 561nm and 640 nm were used to excite AcGFP1/QDs, mCherry/TMR and Dylight 649/CellMask stain, respectively. The fluorescence signals were collected through 525/50 nm, 605/20 nm and 685/40 nm band-pass filters (Chroma).

Image Analysis The trajectories of virus and time courses of fluorescence intensity were reconstructed from raw images by aligning the coordinates and intensity of spots representing virus and corresponding cellular endocytic structures in each frame with Imaging-Pro-Plus software (Media Cybernetics). Only the integral trajectories within the focal plane were processed in quantitative single-virus tracking analysis. The MSD was calculated with Matlab software (MathWorks). The different modes of diffusion were determined by fitting the dependence of MSD against time with the functions as below.47 MSD = 4Dt

normal diffusion (1)

MSD = 4Dtα

restricted diffusion (2)

MSD = 4Dt + (Vt)2

directed diffusion (3)

D represents the diffusion coefficient and V represents the mean velocity. In equation 2, α

Real-Time Dissection of Distinct Dynamin ... - ACS Publications

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