αB-crystallin dependent cell characteristics in rat L6 myoblast cells.

<p>αB-crystallin dependent cell characteristics in rat L6 myoblast cells.</p

Effect of paclitaxel on αB-crystallin-overexpressing C6 cells and knockdown cells.

<p>Cell shape change after paclitaxel treatment. Just after the addition of drug (final 20 μM; 0 min, left panel) and 90 min later (right panel) (A). Cell area (B) and Shape index (C) change of C6 cells after paclitaxel treatment (final 20 μM). N = 20.</p

Effect of cytochalasin D on C6 αB-crystallin-overexpressing and knockdown cells.

<p>Just after drug addition (final 0.4 μM; 0 min, left panel) and 45 min (right panel) (A). Cell area (B) and Shape index (C) change of C6 cells after Cytochalasin D (final 0.4 μM). N = 20.</p

Average migration speed of cells.

<p>Fast migrating leukocytes (~300–1500 μm/hr.) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref037" target="_blank">37</a>]; single motile breast cancer cells (180 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref038" target="_blank">38</a>]; neuron migration in developing cerebral cortex (~60 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref039" target="_blank">39</a>]; normal human epidermal keratinocytes (58.2±2.4 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref040" target="_blank">40</a>]; migratory somatic cells (37±15 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref041" target="_blank">41</a>]; αB-crystallin knockdown C6 glioma cells (40.1 μm/hr, this study); αB-crystallin knockdown L6 myoblast cells (20.2 μm/hr, this study); mouse E9.0 primordial germ cells (16.2±2.5 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref042" target="_blank">42</a>]; primary human dermal fibroblasts (15 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref043" target="_blank">43</a>]; astrocytes (15 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref044" target="_blank">44</a>]; primary human myoblasts (10.5±5.8 μm/hr) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168136#pone.0168136.ref045" target="_blank">45</a>]; C6 glioma cell (12.1 μm/hr, this study); L6 myoblast cells (7.2 μm/hr, this study); αB-crystallin-overexpressing C6 glioma cells (8.5 μm/hr, this study); αB-crystallin-overexpressing L6 myoblast cells (5.8 μm/hr, this study).</p

Mode of migration of wild-type, αB-crystallin overexpressing and knockdown cells.

<p>Bar is 50 μm. Time-lapse images were recorded for 2 h at 10 min intervals and traces of the migration were drawn on the right hand graph. Circles on the left panel indicate the center of the cells at the beginning of the image recordings.</p

Focal adhesion-stress fiber phenotype is αB-crystallin dependent.

<p>Wild-type, αB-crystallin overexpressed, and knockdown L6 cells were visualized for F-actin (green), vinculin (red), and nucleus (blue). Bar is 20 μm.</p

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

<div><p>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.</p></div

Morphology-dependent cell proliferation study of αB-crystallin-overexpressing cells and knockdown C6 cells.

<p>See detail in the text. Bar = 40 μm in A.</p

Cell morphology and αB-crystallin level.

<p>(A) Characteristic cell morphology is specific to αB-crystallin level as revealed by inhibitory antibody injection and (B) injection of purified bovine lens protein αB-crystallin into wild-type, αB-crystallin-overexpressing and knockdown C6 cells. Asterisk indicates P value of <0.05.</p

Impact of Rac1 on morphology of αB-crystallin-overexpressing and knockdown cells.

<p>C6 cells were transfected with EGFP-Rac1 (wild-type), EGFP-constitutively active Rac1 (V12), and EGFP-dominant-negative Rac1 (N17), and fluorescence images were taken after 12 h. αB-crystallin knockdown C6 cells that expressed Rac 1^V12 showed enlarged peripheral ruffles at both ends of spindle-shaped cells. On the other hand, complete loss of lengthened pseudopods was observed in Rac1^N17 transfected cells (A). Area change (B) and shape index (C) of EGFP-Rac1, Rac1^V12, Rac1^N17 expressed wild-type, αB-crystallin overexpressed, and knockdown C6 cells. ** = P<0.01, *P = 0.05, n = 50.</p