Permeabilization-Tolerant Plasma Membrane ... - ACS Publications

Subscriber access provided by University of Pennsylvania Libraries

Article

Permeabilization-Tolerant Plasma Membrane Imaging Reagent Based on Amine-Rich Glycol Chitosan Derivatives Hong-Yin Wang, Jie Sun, Liu-Yuan Xia, Yan-Hong Li, Zhan Chen, and Fu-Gen Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00448 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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 free 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 accessible to all readers and 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.

ACS Biomaterials Science & Engineering 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 30

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 Biomaterials Science & Engineering

Permeabilization-Tolerant Plasma Membrane Imaging Reagent Based on Amine-Rich Glycol Chitosan Derivatives

Hong-Yin Wang,† Jie Sun,† Liu-Yuan Xia,† Yan-Hong Li,† Zhan Chen,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan *Corresponding Author: E-mail: [email protected]

1

ACS Paragon Plus Environment


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 30

ABSTRACT Immunofluorescence staining is a crucial tool for studying the structure and behavior of intracellular proteins and organelles. During the staining process, the permeabilization treatment is usually required to enhance the penetration of fluorescent antibody into the cells. However, since most of the membrane imaging dyes as well as the membrane lipids will detach from the cell surface after permeabilization, membrane labeling using these dyes is not compatible with immunofluorescence staining. Herein, by linking cholesterol-polyethylene glycol (PEG-Chol) and fluorescein isothiocyanate (FITC) with the amine-rich glycol chitosan (GC), we prepared a multifunctional polymeric construct, GC-PEG Chol-FITC, and realized permeabilization-tolerant plasma membrane imaging. Owning to the presence of abundant amine groups in the labeling reagent and the membrane proteins/lipids, the addition of paraformaldehyde in the fixation step induces the amine-crosslinking between the labeling reagents and the membrane proteins/lipids, thus preventing the detachment of fluorophores from the cell surface after permeabilization. Besides, the large molecular weight effect of the imaging reagent may also account for its anti-permeabilization property. Furthermore, by combining immunofluorescence staining with the plasma membrane labeling by GC-PEG Chol-FITC, we simultaneously imaged plasma membrane and cytoskeletons, and clearly observed metaphase cells and binucleated cells. The concept of using amine-rich polymeric dyes

for

plasma

membrane

imaging

will

inspire

the

development

of

more

permeabilization-resistant membrane labeling dyes with better performance, which can realize simultaneous membrane and intracellular protein imaging and facilitate the future studies of membrane–intracellular protein interactions.

KEYWORDS: immunofluorescence staining, cellular imaging, cell surface engineering, permeabilization, glycol chitosan

2


Page 3 of 30

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


INTRODUCTION Plasma membrane labeling is a powerful tool to study plasma membrane-related cellular behaviors such as cell adhesion, division, endocytosis, apoptosis, and signal transduction. Commonly used commercial fluorescent dyes for membrane imaging include carbocyanines (such as DiD, DiO, and DiI), FM 4-64, CellMask, etc. Recently, with the development of cell surface engineering,1–7 a number of novel membrane probes have been developed to improve the imaging performance, such as extending the retention time of the probes on plasma membrane (with less internalization),8–13 enhancing the probe’s photostability for long-time experimental observation,14,15 providing different colors (blue, far-red/near-infrared, and dual-color) for potential multicolor imaging,16–18 realizing “wash-free” imaging of targeted plasma membrane proteins,19,20 and optimizing membrane affinity by mimicking the structure of natural glycolipids.21 On the other hand, it is desirable to simultaneously stain plasma membrane and various intracellular components including nucleus, cytoplasmic organelles, cytoskeleton, and various proteins of interest so that detailed information about the relative locations and spatial relationships of these intracellular components with the membrane can be acquired. For example, mitosis study usually needs to trace the dynamic changes of nucleus, cytoskeleton, and plasma membrane simultaneously. Technically, nucleus and organelles can be labeled in a live cell using commercial dyes like Hoechst (for nucleus), MitoTracker (for mitochondrion), LysoTracker (for lysosome), and ER-Tracker (for endoplasmic reticulum). However, labeling of cytoskeleton and intracellular proteins mainly depends on immunostaining realized through targeting specific proteins by fluorescence-labeled antibodies. During immunostaining, the

3



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

cells need to be permeabilized using detergents (such as Triton X-100, tween-20, NP-40, and digitonin) to enhance the antibody penetration into the cells. This process also removes the lipids from the cell plasma membrane. Prior to the detergent treatment, the cells should be fixed, usually using 4% paraformaldehyde (PFA), to maintain the structure and the shape. However, after permeabilization, most of the commonly used membrane labeling dyes will detach from the cell surface along with the removed membrane lipids. For instance, the long-chain carbocyanine dye DiI is incompatible with immunofluorescence staining because Triton X-100 treatment can cause diffusion and fading of DiI from the labeled structures.22 Additionally, as described in the product’s protocol, another commercial membrane labeling dye CellMask can only be used under the conditions without detergent treatment. Therefore, to ensure excellent membrane labeling effect and avoid membrane removal during immunofluorescence staining, membrane labeling dyes that can tolerate permeabilization is highly desirable. A few commercially available dyes have been reported to be compatible with permeabilization. CellTracker CM-DiI, a DiI derivative, contains the thiol-reactive chloromethyl moiety that can covalently bind to cellular thiols, enabling this dye to be retained in some cells after permeabilization. For example, CM-DiI has been used to label the plasma membranes of micropatterned osteoblasts, followed by actin labeling through immunofluorescence staining.23 Nevertheless, it was reported in some cases that the permeabilization treatment should be conducted using mild detergents, because some agents such as Triton X-100 could induce loss of CM-DiI labels.24,25 Sulfonated carbocyanine dyes SP-DiIC18(3) and SP-DiOC18(3) are compatible with aldehyde-based fixation and

4


Page 4 of 30

Page 5 of 30

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


acetone-based lipid extraction for certain cell lines. However, the mechanism for the cell retention of these dyes still remains unclear. Wheat germ agglutinin (WGA, a lectin) can specifically bind N-acetylneuraminic acid (sialic acid) residues in glycoproteins found at the membrane of mammalian cells, thus fluorescence-labeled WGA (WGA conjugates), such as WGA-FITC,26 WGA-quantum dots (QDs),27 and WGA-64728 were used to stain the plasma membrane of mammalian cells. Since this specific recognition-based staining method to some extent is independent of lipids (although some sialic acid-containing glycolipids also bind WGA), WGA conjugates are also tolerant to permeabilization, as reported previously.29,30 However, since different cell types may have markedly varied cell surface sugars, the performance of WGA conjugate labeling differs in different cells.8 Although BODIPY TR methyl ester was also reported to be detergent treatment tolerant, this dye readily permeates the plasma membrane and mainly labels endomembranous organelles, not the plasma membrane itself.31 Therefore, for simultaneous plasma membrane labeling and intracellular immunostaining, it is necessary to develop plasma membrane imaging dyes that can tolerate permeabilization. In this work, we discovered that our previously developed multisite plasma membrane imaging reagent, glycol chitosan (GC)-polyethylene glycol (PEG) cholesterol-fluorescein isothiocyanate (FITC) (abbreviated as GC-PEG Chol-FITC,11 with its chemical structure shown in Scheme 1A), could resist lipid removal from cell membrane by Triton X-100 due to the cross-linking of amine groups on the backbone of GC during the aldehyde-based fixation step. As shown in Scheme 1, GC-PEG Chol-FITC could anchor to the plasma membrane via hydrophobic interaction between the cholesterol moieties and the outer layer lipids in the

5



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

membrane. After cell fixation with 4% PFA, the amine-rich GC-PEG Chol-FITC molecules could be crosslinked with each other and also with membrane proteins. Therefore, despite of the possible lipid removal from the cell membrane during permeabilization, the crosslinked GC-PEG Chol-FITC molecules remain on the cell surface, making GC-PEG Chol-FITC a good membrane labeling reagent that can tolerate lipid removal in immunostaining. The concept of using amine-rich polymeric dyes for plasma membrane imaging could inspire the design of more permeabilization-tolerant membrane labeling dyes for simultaneous plasma membrane and intracellular protein imaging, advancing membrane-related studies.

Scheme 1. (A) Chemical structure of the staining reagent, GC-PEG Chol-FITC. (B) Schematic illustration of GC-PEG Chol-FITC which could tolerant the permeabilization treatment. When encountering cells, GC-PEG Chol-FITC could anchor to the plasma membrane via the cholesterol’s membrane anchoring ability. After fixation with 4% PFA, the amine-rich GC-PEG Chol-FITC molecules could be crosslinked with each other and with membrane proteins. Therefore, although lipids could be removed from the plasma membrane by permeabilization, the crosslinked GC-PEG Chol-FITC molecules still remain on the cell surface.

6


Page 6 of 30

Page 7 of 30

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


EXPERIMENTAL SECTION Materials. GC (G7753) was purchased from Sigma-Aldrich (St. Louis, MO). Its molecular weight

(MW) was reported to be 67 kDa (Mn) or 100 kDa (Mw), and the degree of deacetylation was 88%.32 FITC was bought from Fanbo Biochemicals Co., Ltd. (Beijing, China). Avidin was obtained from Sangon Biotech (Shanghai, China). N-Hydroxysuccinimide (NHS) PEG2k-cholesterol (NHS-PEG-Chol),

cholesterol-PEG2k-FITC

(Chol-PEG-FITC)

and

cholesterol-PEG2k-biotin

(Chol-PEG-biotin) were purchased from Nanocs, Inc. (New York, NY). NHS-biotin was produced by J&K Scientific Ltd. (Beijing, China). NHS-PEG8-OMe was purchased from Biomatrik Inc. (Jiaxing, China). Tubulin-Tracker Red (Alexa555-conjugated anti-α-Tubulin antibody) and Hoechst 33342 were ordered from Beyotime Institute of Biotechnology. (Nantong, China). CellMask Green was purchased from Invitrogen (Carlsbad, CA). Avidin-conjugated quantum dots (avidin-QDs, CdSe/ZnS core–shell structure, ~7 nm diameter, maximum emission wavelength 605 nm) were obtained from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), PFA, formaldehyde, ethanol, methanol, and Triton X-100 were bought from Aladdin Reagent Company (Shanghai, China). Deionized water (18.2 MΩ·cm) was obtained from a Milli-Q synthesis system (Millipore, Billerica, MA). Dialysis membranes were purchased from Spectrum Labs (Rancho Dominguez, CA). A549 lung cancer cells were ordered from the American Type Culture Collection (ATCC, Manassas, VA). Synthesis of GC-PEG Chol-FITC. GC-PEG Chol-FITC was synthesized according to our previously reported method9 with minor modification. In brief, 5.0 mg NHS-PEG-Chol and 4.0 mg GC were separately dissolved in 1.0 mL phosphate buffered solution (PBS, pH 7.4, 50 mM) and mixed together

7



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

to react for 4 h under stirring at room temperature. After dialyzing (MWCO 10K) against deionized water for 3 days and lyophilization, the GC-PEG Chol compound was obtained. Then 1.0 mg GC-PEG Chol was dissolved in 1.0 mL sodium carbonate buffer (pH 9.5, 0.1 M) and 16 µL FITC (1 mg/mL in DMF) was added to the solution. The mixture was allowed to react at 4 °C overnight. After dialysis (MWCO 10 kDa) and freeze drying, GC-PEG Chol-FITC reagent was finally obtained. All the procedures were carried out in dark. Successful synthesis of the reagent was verified by 1H NMR (Figure S1). Preparation of Amine-Free GC-PEG Chol-FITC-PEG8. To eliminate the amine groups on GC-PEG Chol-FITC, we directly incubated this dye (1.0 mg) with overdosed NHS-PEG8-OMe (4.0 mg) in PBS (pH 7.4, 50 mM) for 12 h at room temperature. Then, the as-prepared product GC-PEG Chol-FITC-PEG8 was purified via dialysis (MWCO 10 kDa) for 3 days. Synthesis of GC-PEG Chol-Biotin. Typically, 15.7 mg GC and 15.3 mg NHS-PEG-Chol were dissolved in PBS ( pH 7.4, 50 mM) and subsequently mixed with 2.6 mg NHS-biotin (dissolved in 0.4 mL DMSO). The mixture was left to react overnight upon stirring at room temperature. After 3 days of dialysis (MWCO 10 kDa) against deionized water and lyophilization, purified GC-PEG Chol-biotin was obtained. Synthesis of Avidin-FITC. To conjugate FITC to avidin, 0.2 mg FITC in 100 µL DMF and 2 mg avidin in 1.0 mL sodium carbonate buffer (pH 9.5, 0.1 M) were mixed together and stirred at 4°C overnight. The resulting avidin-FITC was purified by dialysis (MWCO 10 kDa) and freeze-dried for further use. Cell Staining, Fixation, and Permeabilization. A549 cells were seeded at 5 × 104 cells/well in 35 mm confocal dishes and cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with

8


Page 8 of 30

Page 9 of 30

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


10% fetal bovine serum (FBS) and 100 IU/mL penicillin–streptomycin at 37 °C with 5% CO2. After culturing for one day when the cells grew to 80% confluence, the cells were washed with PBS twice and stained with various plasma membrane staining dyes prepared in DMEM. For one-step staining, the cells were incubated with Chol-PEG-FITC (10 µg/mL), CellMask (1:1000 dilution), or GC-PEG Chol-FITC (100 µg/mL) at 37 °C for 10 min, respectively. For two-step staining, the cells were first incubated with Chol-PEG-biotin (10 µg/mL) or GC-PEG Chol-biotin (100 µg/mL) at 37 °C for 10 min to biotinylate cell surfaces. After washing with PBS twice, the biotinylated cells were stained with avidin-QDs (20 nM) or avidin-FITC (20 µg/mL) at 37 °C for 10 min. For cell fixation, the plasma membrane-labeled cells were first washed with PBS twice and then fixed with 4% PFA, 4% formaldehyde, or 95% ethanol (with 5% acetic acid to counter the shrinkage caused by ethanol) at room temperature for 10 min, respectively. For cell fixation with methanol, the procedure was conducted at –20 °C for 10 min. Cell permeabilization was performed with 0.2% Triton X-100 at room temperature for 10 min followed by PBS washing twice. For reversed staining (staining after fixation) of GC-PEG Chol-FITC, cells were first fixed with 4% PFA , and then stained with GC-PEG Chol-FITC. For reversed staining of avidin-FITC, cells were first incubated with Chol-PEG-biotin. Then biotinylated cells were fixed with 4% PFA followed by staining with avidin-FITC. 4% PFA-Induced Aggregation of GC-PEG Chol-FITC. To test whether PFA could induce crosslinking of GC-PEG Chol-FITC, 50 µL 4% PFA was added to 100 µL GC-PEG Chol-FITC (1 mg/mL) or PBS solution (control) and incubated at room temperature for 12 h. Then the mixtures were centrifuged at 14500 rpm for 10 min, and the formation of sedimentation at the bottom of the Eppendorf tube was examined. Immunofluorescence Staining and Confocal Imaging. After labeling with GC-PEG Chol-FITC (100

9



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

µg/mL) at 37 °C for 10 min, the cells were washed with PBS and fixed with 4% PFA at room temperature for 10 min. Then the cells were treated with 0.2% Triton X-100 in PBS at room temperature for 10 min. After washing with PBS, the cells were incubated with Tubulin-Tracker Red (1 : 250 dilution) and Hoechst 33342 (10 µg/mL) at room temperature for 20 min, followed by PBS washing for twice. Confocal fluorescence images were taken using a confocal microscope TCS SP8 (Leica, Germany) with 63× or 100× oil immersion objectives. The 488 nm laser was selected to excite the FITC-based dyes (Chol-PEG-FITC, GC-PEG Chol-FITC, and avidin-FITC) and the fluorescence signal was detected in the wavelength range of 500–550 nm. Hoechst 33342 was excited with 405 nm laser and the fluorescence signal was detected in the range of 410–500 nm. Avidin-QDs were excited with 488 nm laser and the fluorescence signal was recorded in the range of 550–650 nm.

RESULTS AND DISCUSSION Tolerance of GC-PEG Chol-FITC against Permeabilization. As mentioned above, the commercial membrane labeling dye CellMask could be removed by detergent treatment. As shown in Figure 1A, although this dye could resist 4% PFA fixation, it totally disappeared after the cells were treated with Triton X-100 (Figure 1D). Therefore it is not suitable for intracellular immunofluorescence staining. Next, we evaluated the performance of another membrane labeling molecule, Chol-PEG-FITC, which can effectively stain plasma membrane via the insertion of cholesterol into the lipid membrane.33 Similarly, this dye also experienced the loss of fluorescence signal after permeabilization (Figure 1B and E). This is because Chol-PEG-FITC does not have functional groups such as amine, carbohydrazide, or chloromethyl that can covalently link to the surrounding biomolecules. Thus, most of these

10


Page 10 of 30

Page 11 of 30

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


dyes along with the lipids in the membrane could be removed by the amphiphilic detergent Triton X-100 (Scheme 2), which can dissolve lipid molecules.34

Figure 1. Confocal fluorescence images of CellMask (A and D), Chol-PEG-FITC (B and E), and GC-PEG Chol-FITC (C and F)-stained A549 cells after fixation with 4% PFA (A–C) and subsequent permeabilization with 0.2% Triton X-100 (D–F). The staining concentrations of CellMask, Chol-PEG-FITC, and GC-PEG Chol-FITC were 1 : 1000 dilution, 10 µg/mL, and 100 µg/mL, respectively.

Scheme 2. Schematic illustration of the behavior of Chol-PEG-FITC after membrane staining and subsequent fixation and permeabilization treatments.

By making use of the fixation procedure in which the formaldehyde reacts extensively with amine groups to form methylene bridges, we believe that the amine-containing membrane dyes could tolerate the detergent treatment. During fixation, the added aldehyde molecules (e.g., 4% PFA) could enable the crosslinking of amine-containing dyes and membrane 11



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

proteins, preventing the removal of the dyes from the cell surface by Triton X-100 treatment (Scheme 1). It was reported that the commercial dye FM 1-43FX, an FM 1-43 analog modified with an amine group, is more fixable compared to FM 1-43 (containing no amine groups).35 However, the tolerance of FM 1-43FX against permeabilization is still not satisfied, since the only one amine group in this dye is insufficient for stable fixation. Additionally, an endocytosis labeling probe with several amine groups (i.e., an octapeptide with one cysteine and seven lysines) demonstrates a better performance over FM 1-43FX in terms of membrane retention.35 Since chitosan polymer is rich in amine groups, we believe that chitosan-based imaging reagents would preserve a sufficient amount of labeling dyes on the plasma membrane after fixation and permeabilization. Glycol chitosan (GC), a biocompatible and amine-rich polymer with good water solubility and stability,36,37 was thus selected as the core element to fabricate a novel membrane labeling reagent that could resist detergent treatment. Since the lipophilic cholesterol has an excellent membrane anchoring ability,38 it was introduced into the GC backbone via a hydrophilic PEG segment (PEG-Chol).39 In addition, FITC molecules were linked to the GC backbone, making the resultant GC-PEG Chol-FITC a fluorescent molecule with the same fluorescence property of FITC (Figure S2). Here, the imaging reagent GC-PEG Chol-FITC refers to GC-10% PEG Chol-2% FITC, with 10% and 2% of the amine groups on GC conjugated with NHS-PEG-Chol and FITC, respectively. The number of amine groups on GC is estimated to be ~419, and thus the numbers of Chol-PEG moieties, FITC molecules, and the remaining amine groups on each staining macromolecule are ~42, ~8, and ~369, respectively (see the calculation details in Supporting Information). Upon incubation with the cells, the GC-PEG Chol-FITC molecule could change conformation

12


Page 12 of 30

Page 13 of 30

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 anchor to the plasma membrane through hydrophobic interaction between its lipophilic cholesterol moieties and the membrane lipids (Scheme 1). As expected, after 4% PFA fixation and Triton X-100 treatment, although a slight decrease in fluorescence intensity was observed, the fluorescent reagents remained on the membrane could still illuminate the plasma membrane with strong green florescence (Figure 1C and F), confirming that GC-PEG Chol-FITC could tolerate the permeabilization treatment. Mechanism of Long Membrane Retention Time of GC-PEG Chol-FITC. There are two major mechanisms for cell/protein fixation: protein denaturation/precipitation induced by dehydrants such as organic solvents (methanol, ethanol, and acetone), and cross-linking between proteins and aldehyde-containing fixatives. To verify that the membrane retention of GC-PEG Chol-FITC was achieved through amine-crosslinking in the fixation procedure, other fixatives including the aldehyde-containing fixative formaldehyde (glutaraldehyde was ruled out because it has strong autofluorescence) and the alcohol fixatives including methanol and ethanol were chosen for comparison. We found that 4% formaldehyde-fixed cells could also realize membrane retention after the Triton X-100 treatment (Figure 2A and D), although the imaging performance was not as good as that of 4% PFA. For alcohol fixatives, because of the absence of aldehyde group, it was expected that the tolerance of GC-PEG Chol-FITC against permeabilization might be lost. This assumption was first confirmed in the methanol-treated group. As shown in Figure 2B and E, after methanol fixation (–20°C, 15 min) and Triton X-100 treatment (room temperature, 15 min), the fluorescence signal on the membrane of GC-PEG Chol-FITC-stained cells dramatically declined. However, a small amount of GC-PEG Chol-FITC molecules were still retained on the surface of methanol-fixed

13



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

cells, illuminating the membrane with weak, sparse fluorescence signal (Figure 2E). This phenomenon suggested that in addition to the amine-crosslinking effect, the large molecular weight of GC-PEG Chol-FITC may also contribute to its membrane-retention property, although this contribution is less significant compared to that of the amine-crosslinking. To verify this large molecular weight effect, we used Chol-PEG-FITC with a relatively smaller molecular weight in the experiment, which could be completely removed from the plasma membrane after direct methanol fixation, even without the Triton X-100 treatment (Figure 3B). This was because that besides the effect of cell fixation, methanol itself could also permeabilize the cell membrane, thus leading to the loss of Chol-PEG-FITC from the plasma membrane. In contrast, GC-PEG Chol-FITC not only survived methanol fixation (Figure 2B), but also partially retained on the membrane even after the permeabilization treatment (Figure 2E), demonstrating the large molecular weight effect of GC-PEG Chol-FITC on its membrane retention property.

Figure 2. Confocal fluorescence images of GC-PEG Chol-FITC-stained A549 cells after fixation with 4% formaldehyde, or methanol, or 95% ethanol, respectively (A–C), and the corresponding confocal fluorescence images after further permeabilization with 0.2% Triton X-100 (D–F). The staining concentration of GC-PEG Chol-FITC was 100 µg/mL. 14


Page 14 of 30

Page 15 of 30

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


For the ethanol-treated cells, the large molecular weight effect was more pronounced. It was found that after 95% ethanol (5% acetic acid) fixation, slight cell distortion was observed and thus the fluorescence-stained membrane became thicker (Figure 2C). After subsequent detergent treatment, the fluorescence intensity only showed a slight decrease, leaving most of the GC-PEG Chol-FITC molecules remaining on the membrane or being trapped in the cell (Figure 2F). In contrast, the fluorescence signal of Chol-PEG-FITC-stained cells was completely lost after ethanol treatment (Figure 3C). The results further demonstrated that GC-PEG Chol-FITC with a high molecular weight could stay much easily on the membrane compared to Chol-PEG-FITC with a smaller molecular weight. Taken together, the fixation experiments indicated that both aldehyde-induced amine-crossslinking and large molecular weight effect contributed to the tolerance of permeabilization treatment of GC-PEG Chol-FITC, and the aldehyde-containing fixative is essential for membrane retention of GC-PEG Chol-FITC.

Figure 3. Confocal fluorescence images of Chol-PEG-FITC-stained A549 cells before (A) and after fixation with methanol (B) or 95% ethanol (C), respectively. The staining concentration of Chol-PEG-FITC was 10 µg/mL.

Next, to verify the aldehyde-induced amine-crosslinking, we added 4% PFA to the 1 15



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 16 of 30

mg/mL GC-PEG Chol-FITC solution (1 : 2, vol : vol) in an Eppendorf tube. After incubation for 12 h, the mixture was centrifuged, and the formation of sedimentation was observed at the bottom of the tube (Figure 4A, right, red arrow). In contrast, no sedimentation was formed in the PBS-diluted GC-PEG Chol-FITC solution (Figure 4A, left), confirming that 4% PFA could induce the crosslinking of GC-PEG Chol-FITC.

Figure 4. (A) Photographs of two Eppendorf tubes containing samples after mixing 100 µL of GC-PEG Chol-FITC solution (1 mg/mL) and 50 µL of PBS (left) or after mixing 100 µL of GC-PEG Chol-FITC solution (1 mg/mL) and 50 µL of 4% PFA (right). The two mixtures were first incubated for 12 h, and then centrifuged at 14500 rpm for 10 min. (B) Confocal fluorescence images of A549 cells after 4% PFA fixation and subsequent GC-PEG Chol-FITC (100 µg/mL) staining.

To

further

prove

4%

PFA-induced

amine-crosslinking

is

essential

for

the

anti-permeabilization performance of GC-PEG Chol-FITC, we performed several control experiments. First, instead of staining the cells before fixation (as we performed above), we fixed the cells with 4% PFA and then stained the fixed cells with GC-PEG Chol-FITC. After the treatment, the staining molecules entered into the cytoplasm (Figure 4B), losing the ability to label the plasma membrane. Furthermore, without 4% PFA fixation, we directly treated the GC-PEG Chol-FITC-stained cells with either PBS (Figure 5A) or Triton X-100 (Figure 5B). 16


Page 17 of 30

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 results showed that the fluorescence on the membrane almost totally disappeared from the Triton X-100 permeabilized cells (Figure 5B), further indicating that the 4% PFA fixation is critical for the successful plasma membrane imaging.

Figure 5. Confocal images of GC-PEG Chol-FITC-stained A549 cells after direct incubation with PBS (A and C) or 0.2% Triton X-100 (B and D) for 10 min. The staining concentration of GC-PEG Chol-FITC was 100 µg/mL.

Finally, we deleted the amine groups on GC-PEG Chol-FITC by directly treating the staining reagent with NHS-PEG8-OMe in PBS. It turned out that although the thus-obtained amine-free GC-PEG Chol-FITC-PEG8 molecules can realize membrane labeling and tolerate fixation (Figure 6A), they were almost completely detached from the membrane after Triton X-100 treatment (Figure 6B), suggesting that the membrane labeling reagent without enough amine groups could not realize anti-permeabilization imaging. Therefore, in the immunostaining procedure, to realize permeabilization-tolerant plasma membrane labeling, we need to first stain the plasma membranes with amine-containing GC-PEG Chol-FITC, and then conjugate the membrane-bound GC-PEG Chol-FITC molecules to the membrane proteins via PFA fixation. 17



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 6. Confocal fluorescence images of amine-free GC-PEG Chol-FITC-PEG8-stained and 4% PFA-fixed A549 cells before (A) and after permeabilization (B) with 0.2% Triton X-100. The staining concentration of GC-PEG Chol-FITC was 100 µg/mL.

Simultaneous Imaging of Plasma Membrane and Cytoskeleton. As discussed above, the simultaneous staining of plasma membrane and intracellular proteins requires the membrane labeling dye to resist the removal by permeabilization. To demonstrate the practical applications of the staining dye, we simultaneously labeled nucleus, microtubule, and plasma membrane of A549 cancer cells with DAPI, Alexa555-conjugated anti-α-tubulin, and GC-PEG Chol-FITC, respectively. Results shown in Figure 7 revealed that all the above cellular structures could be clearly visualized. Importantly, plasma membrane (green) and the midbody structure (red) , which is a cytoplasmic bridge between two nascent daughter cells during the final stages of cell division/cytokinesis, could be simultaneously imaged (Figure 7E, red arrow). During cytokinesis, there is a close interaction between the plasma membrane and the midbody structure. For example, the midbody can provide a site of attachment for the furrow plasma membrane before abscission.40 It was also reported that the inner leaflet lipid phosphatidylserine (PS) in the plasma membrane can become more concentrated at the

18


Page 18 of 30

Page 19 of 30

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


midbody and switch to the outer leaflet.41,42 Therefore, GC-PEG Chol-FITC has the potential to be used to investigate the interactions and relationships between plasma membrane and the midbody. Besides, a dividing A549 cell in metaphase (Figure 8A) and a binucleated A549 cell (Figure 8B) with green fluorescence labeled-plasma membrane were recorded. Notably, without GC-PEG Chol-FITC staining, the cell contour could not be clearly identified in the bright field image, especially in the case of metaphase cells (Figure 8A) where the plasma membrane is not located at the boundary of the cytoskeletons (Figure 8B). These results suggested that simultaneous imaging of nucleus, cytoskeleton, and plasma membrane is very helpful to identify the spatial relationships of relevant structures during cell division and GC-PEG Chol-FITC can be used to study membrane dynamics and identify plasma membrane status (for example, whether plasma membranes of the two daughter cells are physically separated or not). Taken together, we proved that GC-PEG Chol-FITC is compatible with immunofluorescence staining, and it could facilitate the studies related to cell membrane–intracelluar protein interactions. Such an amine-rich plasma membrane labeling reagent would also improve the development of novel plasma membrane labeling dyes that could survive permeabilization.

Figure 7. Confocal fluorescence images of A549 cells co-stained with 10 µg/mL of Hoechst (A), 1:250 dilution of Alexa555-conjugated anti-α-tubulin (B), and 100 µg/mL ofGC-PEG

19



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

Chol-FITC (C) realized by combining plasma membrane labeling with immunofluorescence staining. (D) The merged image of all the three channels. (E) The enlarged image of the upper right corner in (D). The red arrow indicates the midbody structure.

Figure 8. Confocal fluorescence images of a dividing A549 cell in metaphase (A) and a binucleated A549 cancer cell (B). Nucleus, microtubule, and plasma membrane of A549 cells were stained with Hoechst (10 µg/mL), Alexa555-conjugated anti-α-tubulin (1:250 dilution), and GC-PEG Chol-FITC (100 µg/mL), respectively.

Other Membrane Retention Dyes. Inspired by the performance of GC-PEG Chol-FITC, we further evaluated other plasma membrane labeling strategies that we previously developed to see their capability for resisting removal by detergent treatment. Because amine-rich fluorescent dyes have the potential to tolerate removal from cell surface, and proteins such as avidin contain several amine groups, we first investigated the avidin-QDs based membrane imaging method that we reported previously.14 In this imaging strategy, the plasma membrane was first biotinylated using Chol-PEG-biotin, after which the biotin molecules could recruit avidin-QDs to realize QDs-labeled plasma membrane. Since the avidin component contains several amine groups, after aldehyde fixation, we found that it could also tolerate Triton X-100 treatment (Figure 9A), making it a good strategy for photostable plasma membrane imaging that is compatible with immunofluorescence staining. As discussed above, Chol-PEG-FITC only could not resist permeabilization (Figure 1B and E). However, by 20


Page 20 of 30

Page 21 of 30

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


changing avidin-QDs with avidin-FITC, the resulting strategy (Chol-PEG-biotin + avidin-FITC) also worked (Figure 9B), demonstrating the essential role of avidin component. Furthermore, we also discovered that the tolerance of permeabilization of this method would be lost when the fixation was conducted with ethanol, or the fixation procedure was performed prior to avidin-FITC incubation (data not shown), further confirming that the aldehyde-induced amine-crosslinking was critical for membrane retention of the membrane labeling agents. Furthermore, we have also verified that our previously reported long-term plasma membrane imaging strategy realized by GC-PEG Chol-biotin and avidin-FITC12 could also resist permeabilization treatment (Figure 9C) due to the amine-rich property of the GC backbone and the avidin molecules.

Figure 9. Confocal fluorescence images of A549 cells after treating with different staining reagents: (A) Chol-PEG-biotin + avidin-QDs. (B) Chol-PEG-biotin + avidin-FITC. (C) GC-PEG Chol-biotin + avidin-FITC. Before imaging, the cells were fixed with 4% PFA and treated with Triton X-100.

In summary, as listed in Table 1, permeabilization-tolerant plasma membrane imaging realized by amine-rich GC-PEG Chol-FITC has several advantages over commercial products. First, the mechanism for membrane retention of GC-PEG Chol-FITC is PFA-induced amine 21



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 22 of 30

crosslinking which can take advantage of the fixation process. While for the commercial dyes SP-DiIC18(3) and SP-DiOC18(3), their permeabilization tolerance mechanism is unknown. Second, GC-PEG Chol-FITC can tolerate harsh detergent (Triton X-100) treatment, while CM-DiI is only compatible with mild detergents (such as digitonin) and SP-DiIC18(3) and SP-DiOC18(3) are limited to specific acetone permeabilization. Furthermore, compared to WGA conjugates, imaging with GC-PEG Chol-FITC is independent of cell surface sugars, and thus satisfying staining performance can be achieved for a variety of cell types. Finally, due to the abundant amine groups on GC-PEG Chol-FITC, the permeabilization tolerant performance surpasses FM 1-43FX/FM 4-64FX containing only one amine group in each molecule. More importantly, as shown above, the amine-crosslinking combined with QDs-labeling can realize permeabilization-tolerant and photostable membrane imaging, which cannot be achieved by other commercial products.

Table 1. Features of Some Commercial Dyes and GC-PEG Chol-FITC in Terms of Permeabilization Tolerance.

Mechanism Detergent Restriction Performance

CellTracker

SP-DiIC18(3)/

WGA

CM-DiI

SP-DiOC18(3)

Conjugates

Chloromethyl/thi ol binding Mild detergent Moderate

FM 4-64FX

Chol-FITC Amine/aldehy

yde

de

crosslinking

crosslinking

No restriction

No restriction

No restriction

Cell

Not

type-dependent

Satisfying

acid binding

Acetone detergent Moderate

GC-PEG

Amine/aldeh

Lectin/sialic

Unknown

FM 1-43FX/

Moderate

It should also be mentioned that, cell membrane modification using a hydrophobic moiety-containing polymer, such as poly(vinyl alcohol)-alkyl (PVA-alkyl), would usually result in aggregation on the plasma membrane.43 In contrast, GC-PEG Chol-FITC showed 22


Page 23 of 30

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


uniform fluorescence signal on the plasma membranes. This is because PVA-alkyl was obtained by directly conjugating the hydrophobic alkyl chains to the PVA polymer. While for our imaging reagent, the hydrophobic cholesterol moieties and the GC polymer were linked through the hydrophilic PEG moieties, which successfully prevented the resulting molecules from forming large aggregates. The hydrodynamic diameter of GC-PEG Chol-FITC measured by dynamic light scattering was 20 nm, which confirmed the absence of large aggregates.11 Besides, as reported in our previous work, GC-PEG Chol-FITC-stained cells showed relatively uniform fluorescence signals on various types of cell membranes.11,39 Moreover, even after incubation with the cells for 6 h, GC-PEG Chol-FITC showed no obvious cellular internalization.11 Taken together, the amine-rich GC-PEG Chol-FITC with the excellent permeabilization-tolerant plasma membrane imaging performance will be beneficial for immunostaining-involving membrane studies.

CONCLUSION In this research, we found that the glycol chitosan-based plasma membrane imaging reagent, GC-PEG

Chol-FITC,

can

tolerate

membrane

permeabilization

during

the

immunofluorescence staining procedure. The amine-rich GC-PEG Chol-FITC reagent could be crosslinked among themselves and with membrane proteins in the PFA fixation procedure, realizing the membrane retention even after the lipid removal by the detergent treatment. Besides, we found that the large molecular weight effect also made contribution to the anti-permeabilization property of this reagent. We have simultaneously imaged plasma membrane and cytoskeletons, and clearly observed midbody structures, metaphase cells, and

23



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

binucleated cells. In light of the performance of GC-PEG Chol-FITC, we found that our previously reported avidin-involved plasma membrane imaging strategies were also compatible with immunofluorescence staining due to the amine-rich and large molecular weight nature of the avidin molecules. The concept of using amine-rich polymeric dyes for plasma membrane imaging which resist the permeabilization could facilitate the development of more plasma membrane labeling dyes with improved staining performance. The present work also provides a solid foundation for future studies on membrane–intracellular protein interactions.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the National High Technology Research & Development Program of China (2015AA020502), National Natural Science Foundation of China (21673037), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. LYX also acknowledges the support from Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (SJLX16_0054).

24


Page 24 of 30

Page 25 of 30

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


REFERENCES (1) Teramura, Y.; Iwata, H. Cell Surface Modification with Polymers for Biomedical Studies. Soft Matter 2010, 6, 1081–1091. (2) Tobinaga, K.; Li, C.; Takeo, M.; Matsuda, M.; Nagai, H.; Niidome, T.; Yamamoto, T.; Kishimura, A.; Mori, T.; Katayama, Y. Rapid and Serum-Insensitive Endocytotic Delivery of Proteins using Biotinylated Polymers Attached via Multivalent Hydrophobic Anchors. J. Controlled Release 2014, 177, 27–33. (3) Takeo, M.; Li, C.; Matsuda, M.; Nagai, H.; Hatanaka, W.; Yamamoto, T.; Kishimura, A.; Mori, T.; Katayama, Y. Optimum Design of Amphiphilic Polymers Bearing Hydrophobic Groups for Both Cell Surface Ligand Presentation and Intercellular Cross-Linking. J. Biomater. Sci., Polym. Ed. 2015, 26, 353–368. (4) Xiong, X. L.; Liu, H. P.; Zhao, Z. L.; Altman, M. B.; Lopez-Colon, D.; Yang, C. Y. J.; Chang, L. J.; Liu, C.; Tan, W. H. DNA Aptamer-Mediated Cell Targeting. Angew. Chem., Int. Ed. 2013, 52, 1472–1476. (5) Qiu, L. P.; Zhang, T.; Jiang, J. H.; Wu, C. C.; Zhu, G. Z.; You, M. X.; Chen, X. G.; Zhang, L. Q.; Cui, C.; Yu, R. Q.; Tan, W. H. Cell Membrane-Anchored Biosensors for Real-Time Monitoring of the Cellular Microenvironment. J. Am. Chem. Soc. 2014, 136, 13090–13093. (6) Jia, H. R.; Jiang, Y. W.; Zhu, Y. X.; Li, Y. H.; Wang, H. Y.; Han, X. F.; Yu, Z. W.; Gu, N.; Liu, P. D.; Chen, Z.; Wu, F. G. Plasma Membrane Activatable Polymeric Nanotheranostics with Self-Enhanced Light-Triggered Photosensitizer Cell Influx for Photodynamic Cancer Therapy. J. Control. Release 2017, 255, 231−241. (7) Jia, H. R.; Zhu, Y. X.; Chen, Z.; Wu, F. G. Cholesterol-Assisted Bacterial Cell Surface Engineering for Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria. ACS Appl. Mater. Interfaces 2017, 9, 15943−15951. 8) Zhang, C. Q.; Jin, S. B.; Yang, K. N.; Xue, X. D.; Li, Z. P.; Jiang, Y. G.; Chen, W. Q.; Dai, L. R.; Zou, G. Z.; Liang, X. J. A Cell Membrane Tracker Based on Restriction of Intramolecular Rotation. ACS Appl. Mater. Interfaces 2014, 6, 8971−8975. (9) Heek, T.; Nikolaus, J.; Schwarzer, R.; Fasting, C.; Welker, P.; Licha. K.; Herrmann, A.; Haag, R. An Amphiphilic Perylene Imido Diester for Selective Cellular Imaging. Bioconjugate 25



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

Chem. 2013, 24, 153–158. (10) Zhang, X. F.; Wang, C.; Jin, L. J.; Han, Z.; Xiao, Y. Photostable Bipolar Fluorescent Probe for Video Tracking Plasma Membranes Related Cellular Processes. ACS Appl. Mater. Interfaces 2014, 6, 12372−12379. (11) Wang, H. Y.; Jia, H. R.; Lu, X. L.; Chen, B.; Zhou, G. X.; He, N. Y.; Chen, Z.; Wu, F. G. Imaging Plasma Membranes without Cellular Internalization: Multisite Membrane Anchoring Reagents Based on Glycol Chitosan Derivatives. J. Mater. Chem. B 2015, 3, 6165−6173. (12) Jia, H. R.; Wang, H. Y.; Yu, Z. W.; Chen, Z.; Wu, F. G. Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy. Bioconjugate Chem. 2016, 27, 782–789. (13) Wang, B.; Zhu, C. L.; Liu, L. B.; Lv, F. T.; Yang, Q.; Wang, S. Synthesis of a New Conjugated Polymer for Cell Membrane Imaging by Using an Intracellular Targeting Strategy. Polym. Chem. 2013, 4, 5212–5215. (14) Wang, H. Y.; Hua, X. W.; Jia, H. R.; Liu, P. D.; Gu, N.; Chen, Z.; Wu F. G. Enhanced Cell Membrane Enrichment and Subsequent Cellular Internalization of Quantum Dots via Cell Surface Engineering: Illuminating Plasma Membranes with Quantum Dots. J. Mater. Chem. B 2016, 4, 834–843. (15) Li, M.; Nie, C. Y.; Feng, L. H.; Yuan, H. X.; Liu, L B.; Lv, F. T.; Wang, S. Conjugated Polymer Nanoparticles for Cell Membrane Imaging. Chem.–Asian J. 2014, 9, 3121–3124. (16) Kreder, R.; Oncul, S.; Kucherak, Q. A.; Pyrshev, K. A.; Real, E.; Mély, Y.; Klymchenko, A. S. Blue Fluorogenic Probes for Cell Plasma Membranes Fill the Gap in Multicolour Imaging. RSC Adv. 2015, 5, 22899−22905. (17) Collot, M.; Kreder, R.; Tatarets, A. L.; Patsenker, L. D.; Mely, Y.; Klymchenko, A. S. Bright Fluorogenic Squaraines with Tuned Cell Entry for Selective Imaging of Plasma Membrane vs. Endoplasmic Reticulum. Chem. Commun. 2015, 51, 17136–17139. (18) Cui, Q. L.; Wang, X. Y.; Yang, Y.; Li, S. L.; Li, L. D.; Wang, S. Binding-Directed Energy Transfer of Conjugated Polymer Materials for Dual-Color Imaging of Cell Membrane. Chem. Mater. 2016, 28, 4661−4669. (19) Prifti, E.; Reymond, L.; Umebayashi, M.; Hovius, R.; Riezman, H.; Johnsson, K. A Fluorogenic Probe for SNAP-Tagged Plasma Membrane Proteins Based on the 26


Page 26 of 30

Page 27 of 30

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


Solvatochromic Molecule Nile Red. ACS Chem. Biol. 2014, 9, 606−612. (20) Karpenko, I. A.; Collot, M.; Richert, L.; Valencia, C.; Villa, P.; Mély, Y.; Hibert, M.; Bonnet, D.; Klymchenko, A. S. Fluorogenic Squaraine Dimers with Polarity-Sensitive Folding as Bright Far-Red Probes for Background-Free Bioimaging. J. Am. Chem. Soc. 2015, 137, 405–412. (21) Redon, S.; Massin, J.; Pouvreau, S.; De Meulenaere, E.; Clays, K.; Queneau, Y.; Andraud, C.; Girard-Egrot, A.; Bretonnière, Y.; Chambert, S. Red Emitting Neutral Fluorescent Glycoconjugates for Membrane Optical Imaging. Bioconjugate Chem. 2014, 25, 773–787. (22) Holmqvist, B. I.; Ostholm, T.; Ekstrom, P. DiI Tracing in Combination with Immunocytochemistry for Analysis of Connectivities and Chemoarchitectonics of Specific Neural Systems in a Teleost, the Atlantic Salmon. J Neurosci. Methods 1992, 42, 45−63. (23) Fu, R. R; Liu, Q. L.; Song, G. B.; Baik, A.; Hu, M.; Sun, S. J.; Guo, X. E.; Long, M.; Huo, B. Spreading Area and Shape Regulate Apoptosis and Differentiation of Osteoblasts. Biomed. Mater. 2013, 8, 055005. (24) Matsubayashi, Y.; Iwai, L.; Kawasaki, H. Fluorescent Double-labeling with Carbocyanine Neuronal Tracing and Immunohistochemistry Using a Cholesterol-Specific Detergent Digitonin. J Neurosci. Methods 2008, 174, 71−81. (25) Chadwick, E. J.; Yang, D. P.; Filbin, M. G.; Mazzola, E.; Sun, Y.; Behar, O.; Pazyra-Murphy, M. F.;Goumnerova, L.; Ligon, K. L.; Stiles, C. D.; Segal. R. A.; A Brain Tumor/Organotypic Slice Co-Culture System for Studying Tumor Microenvironment and Targeted Drug Therapies. J. Vis. Exp. 2015, 105, e53304. (26) Emde, B.; Heinen, A.; Gödecke, A.; Bottermann, K. Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue after Myocardial Infarction. Eur. J. Histochem. 2014, 58, 315–319. (27) Boileau, A. J.; Pearce, R. A.; Czajkowski, C. The Short Splice Variant of the γ2 Subunit Acts as an External Modulator of GABAA Receptor Function. J. Neurosci. 2010, 30, 4895–4903. (28) Gauvin, T. J.; Young, L. E.; Higgs, H. N. The Formin FMNL3 Assembles Plasma Membrane Protrusions That Participate in Cell–Cell Adhesion. Mol. Biol. Cell 2015, 26, 467–477. (29) Hayashi, H.; Yamashita, Y. Role of N-Glycosylation in Cell Surface Expression and Protection against Proteolysis of the Intestinal Anion Exchanger SLC26A3. Am J Physiol Cell 27



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

Physiol. 2012, 302, 781–C795. (30) Zhang, H.; Chen, Y. Q.; Wadham, C.; McCaughan, G. W.; Keane, F. M.; Gorrell, M. D. Dipeptidyl Peptidase 9 Subcellular Localization and a Role in Cell Adhesion Involving Focal Adhesion Kinase and Paxillin. Biochimica et Biophysica Acta 2015, 1853, 470–480. (31) Baird, N. L.; York, J.; Nunberg, J. H. Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication. J. Virol. 2012, 86, 11301–11310. (32) Pereira, P.; Morgado, D.; Crepet, A.; David, L.; Gama, F. M. Glycol Chitosan-Based Nanogel as a Potential Targetable Carrier for siRNA. Macromol. Biosci. 2013, 13, 1369−1378. (33) Chen, X. K.; Zhang, X, D.; Wang, H. Y.; Chen, Z.; Wu, F. G. Subcellular Fate of Fluorescent Cholesterol-Polyethylene Glycol Conjugate: An Excellent Plasma Membrane Imaging Reagent. Langmuir 2016, 32, 10126–10135. (34) Schuck, S.; Honsho, M.; Ekroos, K.; Shevchenko, A.; Simons, K. Resistance of Cell Membranes to Different Detergents. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5795–5800. (35) Revelo, N. H.; Kamin, D.; Truckenbrodt, S.; Wong, A. B.; Reuter-Jessen, K.; Reisinger, E.; Moser, T.; Rizzoli, S. O. A New Probe for Super-Resolution Imaging of Membranes Elucidates Trafficking Pathways. J. Cell Biol. 2014, 205, 591–606. (36) Yhee, J. Y.; Son, S.; Kim, S. H.; Park, K.; Choi, K.; Kwon, I. C. Self-Assembled Glycol Chitosan Nanoparticles for Disease-Specific Theranostics. J. Controlled Release 2014, 193, 202–213. (37) Jiang, Y. W.; Guo, H. Y.; Chen, Z.; Yu, Z. W.; Wang, Z. F.; Wu, F. G. In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives. Langmuir 2016, 32, 6739–6745. (38) Zope, H. R.; Versluis, F.; Ordas, A.; Voskuhl, J.; Spaink, H. P.; Kros, A. In Vitro and In Vivo Supramolecular Modification of Biomembranes Using a Lipidated Coiled-Coil Motif. Angew. Chem. Int. Ed. 2013, 52, 14247–14251. (39) Wang, H. Y.; Hua, X. W.; Jia, H. R.; Li, C. C.; Lin, F. M.; Chen, Z.; Wu, F. G. Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells. ACS Biomater. Sci. Eng. 2016, 2, 987–997. (40) Gould, G. W. Animal Cell Cytokinesis: The Role of Dynamic Changes in the Plasma Membrane Proteome and Lipidome. Semin. Cell Dev. Biol. 2016, 53, 64–73. 28


Page 28 of 30

Page 29 of 30

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


(41) Arai, Y.; Sampaio, J. L.; Wilsch-Bräuninger, M.; Ettinger, A. W.; Haffner, C.; Wieland B. Huttner, W. B. Lipidome of Midbody Released from Neural Stem and Progenitor Cells During Mammalian Cortical Neurogenesis. Front. Cell. Neurosci. 2015, 9, 325. (42) Chai, Y.; Tian, D.; Yang, Y.; Feng, G.; Cheng, Z.; Li, W.; Ou, G. Apoptotic Regulators Promote Cytokinetic Midbody Degradation in C. elegans. J. Cell Biol. 2012, 199, 1047–1055. (43) Inui, O.; Teramura, Y.; Iwata, H. Retention Dynamics of Amphiphilic Polymers PEG-Lipids and PVA-Alkyl on the Cell Surface. ACS Appl. Mater. Interfaces 2010, 2, 1514–1520.

29



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 Graphic

30


Page 30 of 30

Permeabilization-Tolerant Plasma Membrane ... - ACS Publications

Recommend Documents