Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress

Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 μm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary for persistent adhesion.


Introduction
Although αB-crystallin is categorized as a small heat shock protein (HSP) [1], growing evidence shows that αB-crystallin is a protein that is expressed ubiquitously under unstressed

Cell lines
The C6 rat glioma cell line was a generous gift from Dr. T. Iwaki, Kyushu University, Japan [27]. The L6 rat skeletal myoblast cell line was obtained from the American Type Culture Collection. C6 cells were cultured in F10 (Gibco BRL, Rockville, MD), and the other cells were cultured in DMEM (Gibco BRL, Rockville, MD) both supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL, Life Technologies, Inc., Rockville, MD, USA) and 80 μg/mL kanamycin (Meiji Seika, Tokyo, Japan). All cell lines were maintained at subconfluent densities (60-70%) and were incubated at 37˚C in 5% CO 2 in a humidified chamber.

Preparation of cells overexpressing αB-crystallin or in which αB-crystallin was knocked down
Rat αB-crystalline-plasmids were a generous gift from Dr. T. Iwaki, Kyushu University, Japan [27]. αB-crystallin-overexpressing cell lines were prepared by co-transfection of pSV-neo (neomycin resistant) and pRSV-SSE (which includes the αB-crystalline-sense coding region). αB-crystalline knockdown cell lines were obtained by the co-transfection of pSV-neo and pRSV-SAS (which includes an αB-crystallin-antisense coding region). Plasmids (pSV-neo: pRSV-SSE or AS = 1: 10) were introduced into the cells by electroporation using a CUY-21 electroporator (TR-Tech, Tokyo, Japan). For the control (wild-type) cell lines, only the pSVneo plasmid was introduced. Successfully transfected cells were selected by their neomycin resistance by adding Geneticin to the culture media to a final concentration of 400 μg/mL (G-418, Life Technologies, Inc., Rockville, MD, USA). Single clones were picked up after 2 weeks and αB-crystallin

Time-lapse studies
Viable cells were plated on glass-bottomed dishes (Iwaki, Tokyo Japan) and were observed under an inverted fluorescent microscope (Axiovert 135TV, Carl-Zeiss, Oberkochen, Germany) equipped with Plan-Neofluor 10x, 20x and 40x objectives. Time-lapse micrographs were acquired using a Sensys CCD digital camera (Photometrics, Tucson, AZ, USA) attached to the microscope. For extended image capturing, we used a handmade incubation system that maintains cells under appropriate conditions for culture (37˚C, 5% CO 2 ). HEPES 10 mM (pH 7.2) (final) was added to the culture media to maintain neutral pH during observation. Each dish was photographed up to 2 h at 3 min intervals. The time-lapse images were analyzed using IPLab Spectrum software (Scanalytics, Fairfax, VA, USA.). Cell migration speed was calculated as the distance that the center of gravity of the cell contour moved in each time interval. The shape index (SI) was calculated from the cell contour and indicates the deformation of the shape. The SI of a circle is 1. The formula to obtain SI is as following: [SI] = 4πA/P 2 (A: area, P: Perimeter) [28].

Cell proliferation and cell cycle analysis
C6 wild-type, αB-crystalline overexpressing and αB-crystalline knockdown cells were grown on 10 plates. A set of plates was removed from the incubator for either time-lapse observation or ethanol fixation and propidium iodide staining for flow cytometric analysis (EPICS XL, Coulter) at five different time points when the cell density reached 10%, 25%, 50%, 80%, and 95%.

Drug treatment
The following cytoskeletal targeting drugs were used: Nocodazole (10 μg/mL final concentration, Sigma) for tubulin depolymerization, Paclitaxel (20 μM final conc., Sigma) for tubulin stabilization, and cytochalasin-D (CyD, 0.02 μM final conc., GIBCO BRL) for actin depolymerization. Cells were cultured on a microscope stage for 2 h before adding drugs to the medium.

Microinjection
N-terminal (N1) or C-terminal (C1) anti-αB-crystallin antibody and purified non phosphorylated form of αB-crystalline protein from bovine lens were prepared as described previously [11] and those were injected into the cells with an Eppendorf injection system (Transjector #5246, Micro manipulator #5171). Cells were cultured on Cellocate (Eppendorf) grid-etched glass plates for 36 h before injection. N1/C1-terminal antibodies were diluted 100-times in PBS and injected into αB-crystallin-overexpressing C6 cells. Ten pg of purified αB-crystallin protein was injected into a single αB-crystallin knockdown C6 cell. Alexa-Fluor 488(Abs: 495nm, Em: 519 nm; Molecular Probes) in PBS was co-transfected into the cells as an injection marker. After injection, the culture dish was placed in an incubator on an Axiovert 135TV microscope stage, and both translucent and fluorescent time-lapse pictures were recorded.
Student's t-test. Where appropriate, one way analysis of variance (ANOVA) was used to compare multiple groups. A P value of <0.05 was considered statistically significant.

αB-crystallin protein-dependent alteration of cell morphology and other characteristics
There is growing evidence that αB-crystallin associates with cytoskeletal fibers in dynamic assembly studies using purified proteins [11,29,30] as well as in cell lines [11,19,31] and primary glial cells (S1 Fig). Therefore, we first focused on cell shape to see if there were αB-crystallin-dependent morphological alterations. Both αB-crystallin-overexpressing and knockdown cell lines were prepared and analyzed (S2 Fig). Compared with wild-type cells, αB-crystallin-overexpressing cells had a more rounded and spread shape (Fig 1). Since αAand αB-crystallin has been well known for affecting cell proliferation kinetics in lens epithelial cells [32], cell doubling time was examined in our experiment because microtubule stability may have altered the timing of cell cycle phase therefore proliferation. Data indicated about two-to three-fold longer for αB-crystallin-overexpressing cells: 52.2 hr for C6 (24.3 hr for wild-type C6), and 84.2 hr for L6 (23.8 hr for wild-type L6). Both C6 and L6 αB-crystallinoverexpressing cells had wide filopodia and highly-developed ruffled membranes and therefore cells spread extensively, revealing the presence of stabilized microtubules (S3 Fig). αBcrystallin knockdown cells showed a characteristic shape. Typically, cells possessed a narrow, fibroblast-like shape with extensively lengthened pseudopodia (Fig 1 and S3 Fig). Phenotype of cells overexpressing αB-crystallin or lacking αB-crystallin was morphologically reversible To confirm that the characteristic cell morphology was specific to αB-crystallin, anti-αB-crystallin antibody was introduced into αB-crystallin overexpressing C6 cells by a microinjection technique. Antibodies raised against both N-and C-terminal αB-crystallin protein were injected into overexpressing cells. We found that targeted inhibition of the specific protein showed a knockdown-like phenotype (S5 Fig). Antibody-injected overexpressing cells showed a significantly reduced cell area (P<0.05) (Fig 2A), and in particular, cells injected with the Nterminal antibody showed loss of the spreading phenotype and adopted a fibroblast-like morphology after five h (S5 Fig). On the other hand, C-terminal antibody-injected overexpressing cells possessed lengthened pseudopodia (S5 Fig). Purified bovine lens αB-crystallin proteininjected knockdown cells became phenotypically wild-type (S5 Fig); that is, we observed a significant cell area increment ( Fig 2B) and recovery of the spreading phenotype (asymmetric ruffled-membrane production) (S5 Fig). In summary, the extended radial morphology produced by αB-crystallin overexpression was reversed to that of wild-type by αB-crystallin antibody injection and elongated fibroblast-like morphology due to knockdown was reverted to wildtype, although it involved a multi-step cytoskeletal rearrangement by αB-crystallin protein injection. Thus, the alteration of cell morphology in both overexpressing and knockdown cells was αB-crystallin-specific.

Characteristics of cell populations and their proliferation
To gain insight into αB-crystallin-dependent cell morphology and function, the cells' proliferative characteristics were examined at different cell densities. Phase-contrast live images were observed after 36 h or 48 h of growth for C6 wild-type, αB-crystallin-overexpressing cells and αB-crystallin knockdown cells after they were seeded at 10% confluence on culture dishes. In  are thick (seen as dark shadow). In αB-crystallin-overexpressing cells, lamellipodia and multinucleated cells (identified by arrows) were characteristic. Once the cells divided, they became fused into multinucleated cells (four nuclei maximum). In αB-crystallin knockdown cells at 36  αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress detached from the substrate and most of the trypsin-treated cells could not attach to the dish, floating in the culture medium where they eventually died. This response of knockdown cells is explained by the fact that they could only adhere to the substrate at the tips of the spindleshape structures. When cells were treated with trypsin, the cell body detached easily but not the tips. Abnormal cytokinesis was also observed. Interestingly, active exocytosis was observed at the tips (data not shown). Cell cycle analysis of C6 cells was performed using flow cytometry. The tendency of the right shift of S-phase cells indicated a delay of DNA synthesis in knockdown cells (S6 Fig). The number of cells either at G 0 /G 1 -phase, S-phase, or M-phase was compared at five different cell density points (Fig 3B). Over a rather broad range of cell densities (25%, 50%, and 80%), αB-crystallin-overexpressing cells showed a higher proportion of M phase cells, which may explain the presence of multinucleated cells observed in Fig 3A. At 10%, 25% and 50% cell densities, the majority of the knockdown cell population was at the G 0 / G 1 -phase, consistent with a delayed M phase (S6 Fig).

Cell shape changed by drug treatment
Adaptation to the environment is an essential characteristic of the cell for survival. Among the many factors involved in the process, a balanced and coordinated actin-tubulin system is key for cellular homeostasis. Previously we found that αB-crystallin is a molecular chaperon for tubulin/microtubules through MAPs. We next assessed the level of αB-crystallin expression and its impact on cell shape by treating cells with drugs targeting cytoskeletal elements to alter their morphology. C6 glioma cells are differentiated malignant cells from the brain (neuro-epithelial origin) and are susceptible to various drug treatments. Nocodazole treatment inhibits polymerization of tubulin in the cell, and knockdown cells showed dramatic changes in both cell area and shape. This was consistent with the notion of its protective role ( Fig 4A) (Fujita et al., 2004). Interestingly Nocodazole-treated αB-crystallin-overexpressing cells showed a reduced cell area compared to non-treated cells and they became compacted (Fig 4B and 4C). This can be attributed to an accumulation of Nocodazole-decorated tubulin/microtubules in the cell due to accelerated microtubule dynamics with a higher level of αB-crystallin compared to wild-type C6 glioma cells.
The susceptibility of glioma cells to anticancer drugs is important information for neoplastic treatment. Induced neural differentiation of paclitaxel-treated C6 glioma cells through cell morphologic changes with expression of neural markers was recently reported [33]. Extended morphology of αB-crystallin-overexpressing cells but not αB-crystallin knockdown cells was dramatically reduced by a microtubule stabilization agent, paclitaxel, indicating that stretched morphology is dependent on αB-crystallin-assisted microtubule dynamic instability (Fig 5). Elongated cell rods of knockdown cells were also found to be dynamic-microtubule-dependent ( Fig 5C).
Cytochalasin D inhibits actin polymerization. Kobayshi reported that Cytochalasin D treatment of C6 glioma cells induced an arborized phenotype through formation of spider-like actin rearrangement [31]. As shown in Fig 6, both wild-type and αB-crystallin-overexpressing cells showed a similar spider-like phenotype (Fig 6A) and cell area reduction (Fig 6B). Morphological changes of overexpressing cells were more dramatic than wild-type, consistent with the involvement of αB-crystallin in actin polymerization as reported previously [27].
Cell attachment area and morphology were Rac1-dependent in αBcrystallin-deficient cells expression were examined. All Rac1 V12 -expressing cells showed ruffled membranes that were typical for Rac1 signaling (Fig 7). In the case of αB-crystallin knockdown cells, Rac1 V12 αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress expression doubled the cell attachment area, and this was reduced to one-half by Rac1 N17 . Although the exact mechanism by which αB-crystallin affects cell shape is not clear, both active

αB-crystallin knockdown inhibited persistent cell adhesion
Time-lapse images of cells in which αB-crystallin was overexpressed (left column) or knocked down (right column) were recorded at 0 h, 1 h and 2 h time points (Fig 8). Typically, both small and extended cells were observed in αB-crystallin-overexpressing populations. Cells with active ruffled membranes and dynamic pseudopodia and pseudo M-phase rounded cells that never divided to restore their shape were characteristic of αB-crystallin-overexpressing cells. In knockdown populations, cells were less spread, with weak contact inhibition and extended pseudopodia. Time-lapse images were recorded for 2 h at 10 min intervals and traces of the migration were drawn on the right hand graph. For this study, we used 10 randomly chosen cells from each cell line (except for those cells that moved out of the frame within the time of observation) (Fig 9). Circles on the left panel indicate the center of the cells at the beginning of the image recordings. Note that the image on the left and graph on the right use different aspect ratios. Migration speed and distance were measured, and the correlation between them was plotted (Fig 10). For both myoblast cells (Fig 10A) and glioma cells (Fig 10B), knocked αB-crystallin regulates cell polarity and adhesion stability through altered vinculin positioning To clarify the mechanism where αB-crystallin is precisely involved for cell shaping and migration, vinculin as the essential component of actin stress fiber-focal adhesion was examined by immunofluorescence. Focal adhesion follows the maturation process from nascent integrinactin interaction to myosin II-mediated focal adhesion maturation through paxillin phosphorylation of FAK [34]. During this process, vinculin which has over 14 binding partners, has to be activated from the auto-inhibitory self-binding state. Vinculin regulates directionality and cell polarity, and the knockdown showed reduced traction force and polarity loss [35]. As shown in Fig 11, well-developed actin stress fibers were confirmed independent of αB-crystallin expression but position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. To gain insight the structure-function correlation regarding αB-crystallin orchestrated focal adhesion, and the mode of knockdown cell migration was carefully examined by time-lapse live cell imaging. For successful cell migration, turnover of the focal adhesion is necessary and microtubule targeting may involve controlling disassembly [36]. As shown in Fig 12, position of nucleus was shifted nearly 10 μm within less than a half hour and consistent with the results as already described. Not leading edge but trailing end dramatically shrinked after the rapid disappearring of the active membrane ruffling where Rac-1 is involved, and the force pushed the nucleus place forward along with the cell body onward. We have been reported αB-crystallin as a molecular chaperon for tubulin/microtubule [11]. Knockdown of αB-crystallin dramatically altered the microtubule localization within the cell and showed a comet-like tail structure behind the nucleus where adhesion site turnover may occur (Fig 12C). Exact mechanism why microtubule is rather thick compare to wild-type are unknown but an assembled mixed microtubule structure formed when cellular equilibrium [αB-crystallin] << [tubulin] ratio may have stabilized the microtubule as previously proposed [29].

Discussion
Migration speeds were compared to the reported values in the literature (Fig 13). From these results, the αB-crystallin-dependent cell shape alterations were found to dramatically affect the dynamics of cell adhesion. The αB-crystallin-dependent cell characteristics determined in this study are summarized in Tables 1 and 2 for C6 glioma cells and L6 myoblast cells, respectively.
Both overexpression and knockdown of cytoplasmic αB-crystallin led to cell shape alterations and therefore affected the maintenance of adhesion. The fact that the quantity of αBcrystallin clearly altered cell morphology on a culture dish in the absence of severe stress (heat or oxidative stress) indicates that the roles played by αB-crystallin are physiologically relevant for cell structure and function. Cell adhesion was dramatically enhanced by excess αB-crystallin and greatly reduced by lower levels of αB-crystallin. Knock down of αB-crystallin altered cellular migration. This was due to changes in substrate attachment, which may involve transient increases in [Ca 2+ ]i by activation of stretch-responsive channels and/or mechano-sensitive channels on the membrane. It is also possible that disruption of the linkage between microtubule and microfilament systems was affected [53,54,55], leading to disassembly of the adhesion complex through calpain activation [12,56].
The possible involvement of Rac1 in the microtubule-αB-crystallin adhesion complex and the membrane domain is highly plausible. Although αB-crystallin is a soluble protein, increasing evidence has shown that it can function as a membrane-associated protein. For example, it αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress plays the following roles: (1) a Golgi-associated membrane protein in the lens [57], (2) in fiber cell membranes in the lens [58], (3) enhanced mitochondrial association upon oxidative stress [59], (4) ER anchoring [60], (5) stress-induced αB-crystallin-Ser59 in the plasma membrane of dendrites [61], (6) exosomes [62] and (7) protection against mechanical stress-induced calpain-dependent degradation of FAK in cardiomyocytes [12]. Note that Pereira et. al. have not mentioned changes in cell shape [12], the elongated morphology of αB-crystallin-depleted cardiomyocytes following siRNA treatment is consistent with our results.
Recently, plectin-deficiency has been examined for its role in contractile forces generated by cell adhesion and cell motility both in myoblasts and keratinocytes [63]. The adhesion strength and traction force were weaker for plectin -/myoblast cells than wild-type cells. Nonetheless, the shape of the plectin -/myoblast cells was very similar to αB-crystallin knockdown cells, and the migration speed was not altered. Thus, plectin is not essential for the dynamic behavior of the cells. We showed that a constitutively active form of Rac1 (Rac V12 ) rescued the cell area knockdown phenotype of αB-crystallin, perhaps through FAK localization to lamellipodia [64]. Tubulin is a soluble protein and important for cell division and cell morphology. It also associates with membranes under both normal physiological conditions and stress conditions. Furthermore, it associates with the nuclear membrane [65], affects membrane and organelle dynamics with microtubule-associated motor proteins [66], alters mitochondrial function [67] [68] and is involved in stretch activation of regulatory subunits of NOX2 on transverse tubule (T-T) membranes of the heart muscle [69]. In addition, it has long been known that Rac1 functions with microtubules [70] [71], that membrane-associated Rac1 is a regulatory component of the NOX2 complex [72] and αB-crystallin participates in stretchinduced muscle contraction [12]. Thus, there is much more to be learned about αB-crystallin [1] [73] and its role in microtubule dynamics, mechano-biology and membrane physiology. Finally, we also found that αB-crystallin regulates cell polarity and adhesion stability through altered vinculin positioning. Knockdown of αB-crystallin dramatically altered the microtubule localization within the cell and affected the focal adhesion turnover leads to unstable adhesion. This study showed a direct evidence of a small heat shock protein, αB-crystallin, is not only important for health and diseases but also showed the protein is a part of the large complex of focal adhesion and play a key role in cell migration. In wild-type (WT), overexpressing (OE) and knockdown (KD) C6 glioma cells and L6 myoblast cells were analyzed by Western blotting using αB-crystallin antibody. After the primary antibody (anti-αB C1 rabbit polyclonal antibody) treatment [11,19], blots were incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) and signals were detected using the ECL system (Amersham Biosciences UK). The result of densitometry analysis shows these differences in the expression levels of αB-crystallin, where wild-type is 1.