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Aggregation induced emission fluorogens light cells via microtubules: accessing the mechanisms of intracellular trafficking of ionic substances Shixin Zhou, Yijun Xia, Yinan Liu, Qihua He, and Bo Song Langmuir, Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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Aggregation induced emission fluorogens light cells via microtubules: accessing the mechanisms of intracellular trafficking of ionic substances

Shixin Zhoua, Yijun Xiab, Yinan Liua, Qihua Hec,* and Bo Songb,*

a

Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, P. R. China b

Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China

c

Center of Medical and Health Analysis, Peking University Health Science Center, Beijing 100191, P. R. China

*

Corresponding author Prof. Dr. Bo Song Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China E-mail: [email protected]; Fax: +86-512-65883097; Tel: +86-512-65882507

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Abstract Understanding the enrichment and intracellular trafficking of substances is centrally important to the biological systems. Here, employing an amphiphilic molecule (denoted by TPE-11) bearing tetraphenylethene moiety, known for aggregation induced emission property, we demonstrated its localization shifting in Hela cells after prolonged incubation. Through a set of delicately designed experiments, we found that one type of cytoskeleton, i.e. microtubule, is responsible for the intracellular transportation regardless of the sources of fluorogens, via endocytosis pathways or not. As the polymerization of microtubules was blocked, the TPE-11 fluorogens were hindered to move to the inner cytoplasma, but scattered in the cells. On the contrary, blocking the polymerization of microfilament has no such effect. We assume that the dynamic polymerization of microtubules should be responsible to the transportation of TPE-11. More importantly, we found that the interaction between TPE-11 and microtubule proteins also happens during process of polymerization in vitro. The intracellular trafficking of TPE-11 by microtubules may be generalized to other amphiphilic molecules as well as endocytosis pathway, and serves as references in designing functional molecules involved in the intracellular transportation.

Keywords: AIE fluorogens, cell imaging, TPE-11, microtubule, intracellular trafficking

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1. Introduction The eukaryotic cell membrane, which consists of the phospholipid bilayer, is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells.1-2 Nano-fluorogens made of organic molecules can interact with the phospholipid bilayer and enter cells.3-8 Some small molecules, such as ions, glucose and amino acids, or specific channels, can directly pass the cell membrane by active and passive diffusion.9 Some larger molecules enter cells via vesicles,

which

involve

in

several

endocytosis

pathways,

including

the

clathrin-mediated, caveolae-mediated, and lipid raft-mediated endocytosis and phagocytosis, as well as pinocytosis and macropinocytosis.10-11. These processes are called internalization,10

In contrast, some inorganic nano-fluorogens (such as

quantum dots) require binding certain specific peptide fragments to obtain high efficient internalization into cells, such as cholera toxin, TAT-peptide, RGD, or phospholipids.5, 12-19 Previously, we reported a novel ionic bolaamphiphile (denoted by TPE-11) bearing tetraphenylethylene, an aromatic moiety with aggregation induced emission (AIE) effect.20-24 Owing to the amphiphilic feature and AIE effect, TPE-11 forms nano-fluorogens with high fluorescence quantum yield, as the concentration is higher than the critical micelle concentration (CMC).20, 25-26 These nano-fluorogens formed at high concentration can easily enter into cytoplasm of Hela cells, and give strong fluorescence mapping of cells.27-35 Our recent results indicate that even when the concentration of TPE-11 is very low (below or at the vicinity of the CMC), where the nano-fluorogens are not formed yet and the fluorescence is supposed to be very weak, the high contrast images can still be obtained. A possible hint is that the local concentration of TPE-11 inside of the cells should be higher than outside of the cell and enough to form micellar aggregates. If this is true, then how the nano-fluorogens are transferred in the inner cells and caused the concentration difference inner / outer of membrane after being internalized into cells. These issues are poorly understood 3


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currently. The cytoplasm of eukaryotic cell is a very complicated system and contains a large variety of molecules (e.g. proteins, RNA, and ions) and organelles.36-41. Within the cytoplasm of eukaryotic cells, materials are mainly transported via two cytoskeletons: microtubule and microfilament.42-45 We hypothesize that the high local concentration of TPE-11 inside of the cells may be correlated with the transportation function of one of these two cytoskeletons. Both cytoskeletons undergo dynamical switch between growing and shrinking phases, resulting in the coexistence of polymerization and depolymerization at the ends.42, 46-47 Herein, a set of experiments were designed to investigate the fluorescence intensity and mapping by blocking or promoting the polymerization reaction of the cytoskeletons using their specific inhibitors and activators (agonists). The results indicate that the TPE-11 molecules (or possible assemblies) were transported to the inner cytoplasm via the dynamic polymerization of microtubules. This line of research demonstrated a general example for the intracellular transportation of amphiphilic molecules, and may be extended to other analogous molecules. 2. Materials and methods 2.1. Staining Hela cells with TPE-11 The Hela cells were cultivated in RPMI 1640 containing 10% fetal bovine serum (FBS, Hyclone). The cells were seeded on coverslips (IWAKI PLL-coated cover glass, ASAHI Techno, Japan) individually placed in a 6-well plate. When the cells became adhesive to the coverslips, they were treated with TPE-11 [final concentration 2.0 µg mL-1, i.e. 1.6 µM]. After the cells being cultivated for defined time, the cultural mediums were replaced by fresh mediums without TPE-11. The coverslips with cells were taken out, and carefully flushed twice with phosphate buffered saline (PBS). The Hela cells were fixed by immersing the coverslips in polyformaldehyde [1%] for 20 min at ambient conditions, and then treated with Triton-100 [0.05%] for 30 min. 2.2. Staining cytoskeleton (microfilament and microtubule) 4


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Prior to staining the cytoskeletons, the above-processed Hela cells were sealed with 2% bovine serum albumin (BSA) for 1 h. The methods to stain microfilament and microtubule are different. (1) Staining microfilament. Herein the microfilaments refer to F-actin. The staining was conducted by incubating the coverslips with Hela cells in phalloidin-TRITC (the as received solution was diluted for 1000 times, Beyotime, China) at 37 °C for 1 h. (2) Staining microtubules. The microtubules refer to β-tubulin. During the staining, the coverslips with Hela cells firstly incubated in antibody [D66] (the as received solution was diluted for 1000 times, ab11307, Abcam, USA) at 37 °C for 1 h; then washed with PBS three times and incubated in second antibody, goat anti-mouse Alexa Fluor647 (Beyotime, China) for 40 min at ambient conditions. After the cytoskeletons being stained, the cells were washed with PBS twice. The coverslips with adhesive Hela cells were then taken out, sealed with glycerol and observed under confocal laser scanning microscopy (CLSM, LEICA STP6000, Germany). 2.3. Fluorescence-activated cell sorting (FACS) analysis The flow cytometry used for FACS analysis was Gallios machine (Beckman Coulter, USA). The excitation wavelength was 405 nm, and the wavelength of bandpass filtering was 550 ± 20 nm. Before the measurement, the Hela cells with different incubation times in TPE-11 were detached from dishes by trypsin, collected in tubes, and then washed with PBS containing 0.5% of FBS twice. The fluorescence signal and counts were acquired from approximately 10,000 events on the flow cytometry, and analyzed using Flowjo 7.6 tools. 2.4. Inhibitors and agonists of cytoskeleton Hela cells were seeded on coverslips and cultivated over night until they were adhesive to coverslips. Inhibitors and agonists of cytoskeleton were added to the culture mediums with final concentrations of 5.0 µM for Paclitaxel (Taxol) and Colchicine, 0.1 µM for Phalloidin, 30 µM for Cytochalasin B and 1.0 µM for EDTA. Paclitaxel and Phalloidin were bought from Sigma-Aldrich, USA. Colchicine and

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Cytochalasin B were acquired from Beyotime, China. EDTA was purchased from Ambion, Life Tecnlologies, USA. 2.5. Polymerization of microtubules and fluorescent signaling generated by TPE-11 in vitro Microtubule polymerization in solution was performed according to the manufacturer’s protocols of tubulin polymerization assay kit (Cytoskeleton inc, USA). Briefly, a final concentration of 3.0 mg mL-1 tubulin was added to the reaction buffer containing 80 mM of PIPES [pH 6.9], 2 mM of MgCl2, 0.5 mM of EGTA, 1 mM of GTP and 10.2% of glycerol. We designed 5 groups of experiments, low concentration (8 µg mL-1) of TPE-11 (G1), High concentration (50 µg mL-1) of TPE-11 (G2), only tubulin (G3), low concentration (8 µg mL-1) of TPE-11 with tubulin (G4) and high concentration (50 µg mL-1) of TPE-11 with tubulin (G5). The TPE-11 solutions with defined concentrations were added to the wells in 96-well plate and the total volume was 100 µL. Then the mixtures were incubated at 37 °C for 3 min and quickly transferred the 96-well plate to the micro-plate reader. The optical density (OD) values of 96-well plate were read by micro-plate reader (Multiskan MK3, Thermo Scientific, USA) at 405 nm every 3 min. 3. Results and discussion 3.1. Fluorescent intensities enhanced with prolonging treatment time of TPE-11 During the experiment, the concentration of TPE-11 applied to stain the Hela cells was 2.0 µg mL-1, at the vicinity of the CMC of TPE-11 in aqueous solution, where TPE-11 is supposed to disperse in water as individual molecules or oligomers. The high contrast fluorescence images (i.e. strong fluorescence emission) suggest the formation of adequate self-assemblies of TPE-11. In addition, the contrast of the fluorescent images enhances with prolonged cultivation time. Herein, the cultivation time after addition of TPE-11 (the details of treatment, collection and detection of the cells are shown in the experimental section) varied from 1, 3, 6 to 12 h. The analysis on data of flow cytometry is based on the comparison of the test group (treated with 6


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TPE-11) and the control (untreated) group (Fig. 1a). As shown in Fig. 1, the boundary of positive and negative signals is about 103 (logarithmic scales) of fluorescent intensities. The positive rates of TPE-11-treated cells for 1, 3, 6 and 12 h were 69.4%, 98.4%, 98.1% and 98.0%, respectively, whereas the rate of the untreated control was 1.4%. The peaks of fluorescent intensity were shifted to the right (getting stronger) when the treatment time prolonged (Fig. 1b-1f). If we elevate the boundary to ~ 104 of fluorescent intensities, the positive rates were 22.2%, 80.8%, 86.1% and 87.0% for treatment time of 1, 3, 6 to 12 h, respectively. The positive rate did not improve too much after being treated for 3 h. However, the intensities were getting stronger with increase of treatment time. There is no obvious increase in both positive rates and intensities after being treated for 6 h. This result means prolonging the cultivation time can enhance the fluorescence intensity owing to continuous accumulation of TPE-11 in the cells. As shown above, both the positive rate and intensity reached to equilibrium in 6 h. The cells treated with TPE-11 for 1 and 6 h correspond to distinctively different positive rates and fluorescence intensities. To differentiate these two states, from here on, we denote the cells treated with 1 and 6 h by 1h- and 6h-stage, respectively.

400

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Fig. 1 (a-e) The counts versus fluorescent intensities after different treatment times with TPE-11 analyzed by flow cytometry. The plots display (a) untreated control, and treated with TPE-11 of 2.0 µg mL-1 for (b) 1 h, (c) 3 h, (d) 6 h and (e) 12 h. The fluorescent intensities were displayed as logarithmic scale. The blue and red lines mark the percentages of the positive rates and the percentages for fluorescent 7


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intensities larger than 104, respectively. The excitation wavelength was 405 nm. (f) positive rate versus treatment time.

As aforementioned, the concentration of TPE-11 used in the above experiments was not high enough to give strong fluorescence (lower than the CMC). In fact, the fluorescence of cells was very strong, and became stronger as prolonging the cultivation time. This result suggests that the concentration of TPE-11 molecules inside of the cells must be bigger than that outside of the cells. Prolonging the cultivation time will increase the concentration-difference. It is for sure that the difference was not caused by concentration driven diffusion, but by some active transportation of cells. Then how was this realized, and who carried out the transportation inside of the cells? 3.2. TPE-11 molecules move to internal cytoplasm at 6h-stage In order to figure out the answer to the above questions, we first need to clarify the location of the TPE-11 molecules in the cells at 1h- and 6h-stage. As shown by CLSM in Fig. 2, at 1h-stage, most of the TPE-11 fluorogens (green) co-localize with the microtubules (red); while at 6h-stage, the fluorogens move to internal cytoplasm. We made quantitative measure for localization of two channels (Fig. S1 and Table S1 in supplementary information). Colocalization rate of 1h-stage (40.16%) was slightly decreased compared with that of 6h-stage (33.09%), however, mean intensities of the former (13.68) was lower than those of the latter (23.49). This result showed mean fluorescence intensities of 6h-stage were enhanced comparing with those of 1h-stage by quantitative analysis. The colocalizations of TPE-11 and microtubules indicate these molecules may move to internal cytoplasm along with microtubules. TPE-11 is an amphiphilic molecule bearing two ionic heads, and the assemblies formed by TPE-11 are featured with an ionic surface. This kind of nanoparticles stands for a unique fluorescent material. The transportation of this kind of nanoparticles, to the best of our knowledge, has never been addressed yet. It is known that materials enter cells though either endocytosis pathways or endosomes.48-49 For our case, the fluorogens likely adopted the endocytosis pathway. 8


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To verify this assumption, we used lyso-tracker (red, a marker linked with late endosomes) to label the lysosome, and observed whether they colocalized with TPE-11 (green) or not. It turns out that a part of TPE-11 fluorogens are trafficked in cells via endocytic pathway (Fig. S2 in supplementary information). The endocytosis has been reported to be associated with polymerization of microtubules in plant cells.50 The rest of TPE-11 fluorogens may move into cells by other mechanism, perhaps via cytoskeletons directly. To test this hypothesis, a group of experiments were conducted to find out the mechanisms of the transportation.

Fig. 2 The CLSM images of Hela cells treated with TPE-11. The columns from left to right are TPE-11 signals (green), microtubules (red), and merged channels. The rows refer to samples at (a-c) 1h stage and (d-f) 6h-stages. Scale bars: 25 µm. First, cargo transportation through cytoskeletons needs their partner motor proteins, cytoplasmic dynein, kinesin or myosin.44 Our previous data showed that inhibition of cytoplasmic dynein kept most of TPE-11 fluorogens staying around the cell membrane and could not enter the inner cells (Fig. S3 in supplementary information). This result shows microtubules and the partner cytoplasmic dyneins should participate the transportation of TPE-11 fluorogens and promotion of aggregation, as illustrated in Fig 3a. Inhibition of cytoplasmic dynein (microtubule relative motor protein) can effectively block TPE-11 fluorogens to enter inner cells. This result indicates that microtubules and their motor proteins should be responsible 9


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for the transportation of TPE-11 into cells both in endocytosis-dependent or independent pathways. Then we designed experiment to test the hypothesis that the movement of TPE-11 depends on the polymerization of cytoskeletons. In the following, two procedures were designed to figure out which cytoskeleton was involved in the intercellular transportation of TPE-11. Procedure 1: before treatment by TPE-11, the Hela cells were pretreated with inhibitors or agonists of microtubule and microfilament for 6 h. The function of inhibitors or agonist is to block or promotion of the polymerization of cytoskeletons.47 Procedure 2: after treatment of TPE-11, inhibitors or agonists of microtubule and microfilament were added into the Hela cells in fresh medium and cultivated for 6 h. In both of the procedures, dimethyl sulfoxide (DMSO) was used as vehicle control. These two procedures were illustrated by schemes as shown in Fig. 3b.

Fig. 3. Schematic illustration of the two procedures designed for understanding the mechanisms of intracellular transportation of the TPE-11. Microtubules are composed of heterodimers of α-tubulin (orange small balls) and β-tubulin (blue small balls). Microtubules grow when polymerization of α- and β-tubulin in the “plus end” (near the cell membrane). The cytoplasmic dynein (red and blue cart) on the microtubules carries a cargo (green ball representing for TPE-11) and moves to the inner cytoplasm. 3.3. Fluorescent signaling of TPE-11 in cells significantly declined as the polymerization of microtubule was blocked

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In procedure 1, the inhibitor or agonist was 5 µM of Paclitaxel (Taxol) and Colchicine, 1 µM of EDTA. The signaling was analyzed by CLSM, and the results were presented in Fig. 4. It is clear that the fluorescent intensity of the cells treated with inhibitors of microtubule is weaker than that of the control (Fig. 4a and Fig. S4 in supplementary information). This result indicates that blocking the polymerization of microtubules can inhibit the transportation of the TPE-11 fluorogens. Additionally, without support of the microtubules, the cellular morphology was changed from spindle to round shape. We speculate that the microtubules have a kind of function to enrich the ionic amphiphile TPE-11. Even when the total concentration is low, the local concentration at vicinity of the microtubules is higher enough for TPE-11 to aggregate. This speculation can also be verified by the co-localization of TPE-11 fluorogens and microtubules in DMSO control group. As depicted above, another fact is that adding microtubule stabilizers and destabilizers can remarkably suppress microtubule dynamics and thus reduce the fluorescent signaling of TPE-11. Combining these results, we can conclude that the microtubules should be intermediary agent for transportation of the TPE-11 fluorogens.

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Fig. 4. The CLSM images of Hela cells processed by procedure 1 dealing with the microtubules. The columns from left to right are TPE-11 signals (green), microtubule (red) and merged channels. The rows are samples treated with different agents: (a-c) DMSO, (d-f) colchicine, (g-i) Taxol and (j-l) EDTA. The concentration of TPE-11 used here is 2 µg mL-1. Scale bars: 25 µm. Microfilaments, similar to microtubules, can also polymerize and depolymerize in cells. These processes are controlled by several factors, as introduced in the following. Phalloidin, a bicyclic heptapeptide, binds to actin filaments much more tightly than to actin monomers, and hence can prevent filament depolymerization. Cytochalasin B has opposite function of Phalloidin, and can promote filament depolymerization.51 Using procedure 1, Hela cells were pretreated with Phalloidin (100 nM) and Cytochalasin B (30 µM) for 6 h. Then TPE-11 was added into the medium and 12


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cultivated for another 6 h, and then observed by CLSM.

Fig. 5 The CLSM images of Hela cells processed by procedure 1 dealing with the microfilament. The columns from left to right are TPE-11 signals (green), microfilament (red) and merged channels. The rows are samples treated with different agents: (a-c) DMSO, (d-f) phalloidin and (g-i) cytochalasin B. The concentration of TPE-11 was 2 µg mL-1. Scale bars: 25 µm. As shown in Fig. 5, the fluorescent signals of TPE-11 in the cells treated with DMSO were as bright as those treated with inhibitor or agonist of microfilament. The TPE-11 fluorogens can still move to the inside of the cells. These results indicate that blocking the polymerization of microfilament has little effect on the transportation of the fluorogens. This group of experiment further confirms that the microtubules are responsible for the transportation of ionic fluorogens. Some researcher reported that ATP hydrolysis by actin causes a much less dramatic change in polymer stability relative to GTP in tubulin 47, 52. Presumably, this is a reason that the transportation of TPE-11 is more dependent on dynamic polymerization of microtubules. The 13


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asymmetric alignment of the proteins of microtubules determines that the TPE-11 molecules can only be transported to one direction along microtubules. 3.4. TPE-11 fluorogens can still move in the cells but show disordered distribution as the cells were treated by procedure 2 As depicted in Fig. 3, in procedure 2, the cells were firstly stained with TPE-11, and then the polymerization of microtubules was blocked. By doing this, we can see where the fluorogens will go without polymerization of microtubules. The fluorescent signals were observed by CLSM, and the results are shown in Fig. 6. In this set of experiments, judging from the CLSM images, the fluorescent intensities of TPE-11 look similar for the cells treated with different agents: colchicine, Taxol and EDTA, and were as strong as that of the control group (treated with DMSO). The fluorescent intensities of microtubules with different agents are quite different. The one with Taxol has higher fluorescent intensity compared with that of the control, while the other two images of the cells treated with colchicine and EDTA show much weaker fluorescent intensities. In addition, after being treated with inhibitors or agonist of microtubules, the fluorogens were distributed either at the edge or inside of the cells. These results indicate that the intracellular transportation of the fluorogens mainly relies on dynamic polymerization of microtubules. This explains the messy pattern of fluorescent signals of TPE-11 fluorogens when the process of polymerization and depolymerization are blocked.

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Fig. 6 The CLSM images of Hela cells processed by procedure 2 dealing with microtubules. The columns from left to right are TPE-11 signals (green), microtubule (red) and merged channels. The rows are samples treated with different agents: (a-c) DMSO, (d-f) colchicine, (g-i) Taxol and (j-l) EDTA. The concentration of TPE-11 was 2 µg mL-1. Scale bars: 25 µm.

Analogously, the Hela cells were treated with the inhibitor or agonist of microfilaments, and observed by CLSM. Here the inhibitors were phalloidin and cytochalasin B. As shown in Fig. 7, the TPE-11 fluorogens can still be transported into the inner cytoplasma. These results indicate that blocking the dynamic polymerization has very little effect on the fluorescent signals of TPE-11, as well as 15


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the final locations. That is to say, the microfilaments were not involved in the intracellular transportation of the fluorogens in Hela cells.

Fig. 7. The CLSM images of Hela cells processed by procedure 2 dealing with microfilament. The columns from left to right are TPE-11 signals (green), microtubule (red) and merged channels. The rows are samples treated with different agents: (a-c) DMSO, (d-f) phalloidin and (g-i) cytochalasin B. The concentration of TPE-11 was 2 µg mL-1. Scale bars: 25 µm. We were wondering where TPE-11 fluorogens stay after they get into cells. Our data demonstrated that the majority of fluorescent signals of TPE-11 (green) overlapped with endoplasmic reticulum (ER) when Hela cells were treated with TPE-11 (Fig. S5 in supplementary information). The results suggest that at low concentrations TPE-11 may be transported to ER of cells for long time treatment, thus resulting in high local concentration. This explains the aggregation of TPE-11 molecules at the compartment of cells (e.g. endoplasmic reticulum). That is to say, 16


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microtubules can very possibly be the road for transportation of TPE-11 nano-fluorogens from the cell membrane to ER. 3.5. Fluorescent signals of TPE-11 were enhanced by polymerization of microtubules

in vitro We intend to test whether tubulin polymerization would affect aggregation as well as fluorescence of TPE-11 at low concentration or not in vitro. According to our previous study, when the concentration of TPE-11 is lower than 10 µg mL-1, the molecules can aggregate, but cannot give strong fluorescence in water solution. We use tubulin polymerization assay (Cytoskeleton inc, USA) to simulate the process of microtubule polymerization. This assay contains α-tubulin, β-tubulin, GTP, Mg2+ and reaction buffer, and is used to test whether tubulin polymerization can enhance the fluorescent signals of TPE-11. The concentration of tubulins in this assay is 3 mg mL-1 according to manuals of the manufacturer. We designed 5 experimental groups with the following conditions: G1, low concentration (8 µg mL-1) of TPE-11; G2, high concentration (50 µg mL-1) of TPE-11; G3, only tubulins; G4, low concentration (8 µg mL-1) TPE-11 and then tubulins; and G5, high concentration (50 µg mL-1) TPE-11 and then tubulins. The fluorescence intensities of TPE-11 and microtubule polymerization is presented in Fig. 8. At low concentration without existence of tubulin (G1), the fluorescence intensities of TPE-11 kept low throughout the 57 min processing period. At high concentration without existence of tubulin (G2), the fluorescence intensities of TPE-11 increased rapidly in the early reaction time, then drop down slowly after 20 min. In G3 group, the system contained only tubulins (α-tubulin and β-tubulin). Since the tubulins themselves have fluorescence, thus in comparison with the rest two groups (G4 and G5), G3 was employed as control. G4 and G5 showed higher and highest fluorescence intensities, respectively. These results indicated that fluorescence intensities of TPE-11 were greatly enhanced as the polymerization of tubulin was going on. Therefore, at the in vitro conditions, the dynamic polymerization of microtubule is helpful for the enrichment and aggregation of TPE-11. 17


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G1 G2 G3 G4 G5

0.3

0.2

0.1

0.0 0

20

40

60

Time (min) Fig. 8. The fluorescence intensities of TPE-11 and microtubule polymerization. G1: Low concentration TPE-11; G2:High concentration TPE-11; G3: Only tubulins; G4: Low concentration TPE-11 plus tubulins; G5: High concentration TPE-11 plus tubulins. High concentration TPE-11: 50 µg mL-1; low concentration TPE-11: 8 µg mL-1; Concentration of tubulins: 3 mg mL-1. Interaction time: 57 min. The fluorescence intensities were recorded every 3 min. 3.6. Schematic illustration of the relationship between dynamic microtubule polymerization and transportation of TPE-11 nanoparticles In this study, the lowest concentration investigated in the cell imaging was 2.0 µg mL-1, where TPE-11 tends to form oligomers, i.e. small aggregates. The self-assembled micellar structures are sized in range of tens of nanometers. Comparing to the inner diameter (12 nm) of the microtubules,53 the aggregates formed by TPE-11 are far too bigger to pass through. Therefore, we assume that TPE-11, either monomers or assemblies, are very likely transported from outer surface of the microtubules. And the transportation should be related to the highly ordered subunits and intrinsic polarity of microtubules.39, 45 Based on the above discussions, we propose the transportation process of TPE-11 fluorogens. Perhaps, there are endocytosis-dependent and independent pathways for TPE-11 fluorogens to enter cells. Both ways may be dependent on dynamic polymerization or depolymerization of microtubules. Fluorogens of TPE-11 (via endocytosis or not) are firstly transported into cells through mediation protein (such as motor protein) along polymerization of microtubules. Then, TPE-11 is aggregated and 18


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self-assembled in certain organelles and exhibits fluorescent signal. Inside of the cells, the transportation into the inner cytoplasma is carried out by the polymerization or depolymerization of microtubules. 4. Conclusions Utilizing the unique AIE feature of TPE-11, we have studied the intracellular transportation of ionic fluorogens. Interestingly, as being applied to labeling the Hela cells, TPE-11 gives high fluorescent signal even when the concentration is extremely low. We believe that the concentration of TPE-11 inside of the cells should be higher than that outside of the cells. Presumably, there are endocytosis-dependent and independent pathways for TPE-11 fluorogens to enter cells. As prolonging the cultivation time, fluorescent signals of TPE-11 are enhanced and the fluorogens move from the edge of the cells to inner cytoplasm. A series of experiments were designed to prove that the trafficking of the ionic fluorogens is carried out by the dynamic polymerization or depolymerization of microtubules. Furthermore, the signals of TPE-11 are enhanced in process of tubulin polymerization in vitro. Acknowledgements The authors are great indebt of Dr. Ping Fan for her critical comments and word editing. This research was supported by grants from the National Natural Science Foundation of China (21674075 and 21233003), the Natural Science Foundation of Jiangsu Province (BK20161211), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References 1.

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