Hypertrophy and atrophy inversely regulate Caveolin-3 expression in myoblasts

Caveolin-3 (Cav-3) is a muscle-specific membrane protein crucial for myoblast differentiation, as loss of the protein due to mutations within the gene causes an autosomal dominant form of limb girdle muscular dystrophy 1-c. Here we show that along with p38 activity the PI3-kinase/AKT/mTOR pathway is required for proper Cav-3 up-regulation during muscle differentiation and hypertrophy, as confirmed by the marked increase of Cav-3 expression in hypertrophied C2C12 cells transfected with an activated form of AKT. Accordingly, Cav-3 expression was further increased during hypertrophy of L6C5 myoblasts treated with Arg(8)-vasopressin and in hypertrophic muscles of MLC/mIGF-1 transgenic mice. In contrast, Cav-3 expression was down-regulated in C2C12 myotubes exposed to atrophic stimuli such as starvation or treatment with dexamethasone. This study clearly suggests that Cav-3 expression is causally linked to the maturation of muscle phenotype and it is tightly regulated by hypertrophic and atrophic stimuli.     

Caveolin-3 is a direct molecular partner of the Cav1.1 subunit of the skeletal muscle L-type calcium channel

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C). Retention of LGMD-1C caveolin-3 mutants within the golgi complex.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C).     

Ammonium Increases TRPC1 Expression Via Cav-1/PTEN/AKT/GSK3β Pathway

Hyperammonemia occurring following acute liver failure is the primary cause of hepatic encephalopathy. In the brain, ammonium is catabolised by glutamine synthetase expressed exclusively in astroglia; ammonium overload impairs astroglial homeostatic systems. Previously, we had reported that chronic treatment with 3 mM ammonia increased expression of transient receptor potential canonic 1 (TRPC1) channels and Ca²⁺ release from intracellular Ca²⁺ stores (Liang et al. in Neurochem Res 39:2127–2135, 2014). Glycogen synthase kinase 3β (GSK-3β) has a key role in several astroglial signalling pathways and is known to be affected in various CNS diseases. We have studied the involvement of Cav-1/PTEN/AKT/GSK-3β signalling system in regulation of TRPC1 gene expression by ammonium. Effects of chronic (1–5 days) treatment with ammonium chloride (ammonium), at pathologically relevant concentrations of 1–5 mM were investigated on primary cultures of mouse cerebral astrocytes. We quantified expression of caveolin-1 (Cav-1), membrane content of phosphatase and tensin homologue (PTEN), phosphorylation of AKT and GSK-3β, and expression of TRPC1 channels. Ammonium significantly increased expression of Cav-1 mRNA and protein, mRNA of TRPC1 as well as membrane content of PTEN; conversely phosphorylation of AKT and GSK-3β were significantly decreased. These changes were abolished following astrocytes treatment with siRNA specific to Cav-1, indicating the involvement of Cav-1/PTEN/PI3K/AKT pathway. Similar results were found in the brains of adult mice subjected to intraperitoneal injection of urease (a model for hyperammoniemia) for 1–5 days. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers (used for fluorescence-activated cell sorting of astrocytes vs. neurones) and treated with intraperitoneal injections of urease for 3 days, the Cav-1 gene mRNA expression was up-regulated in astrocytes, but not in neurones. The up-regulation of TRPC1 gene expression by ammonium was suppressed by GSK-3β inhibitors, lithium salt and AR-A014418, suggesting that increase of GSK-3β activity may play a role in ammonium-related pathologies.     

Using Caveolin-1 epithelial immunostaining patterns to stratify human breast cancer patients and to predict the Caveolin-1 (P132L) mutation

Caveolin-1 (Cav-1) mutations, such as P132L, are associated with ER-positive human breast cancers. However, no immuno-histochemical methods have yet been described to predict the presence of Cav-1 mutations in human breast cancer. Since the P132L mutation acts in a dominant-negative fashion and causes the mis-localization Cav-1 in cultured cells in vitro, we hypothesized that patients carrying this mutation would show a similar Cav-1 staining pattern in vivo. Indeed, while performing histological analysis of Cav-1 immunostaining on human breast cancer samples, we noted the emergence of two distinct epithelial staining patterns: 1) punctate peri-nuclear “Golgi-like” localization; or 2) diffuse cytoplasmic staining. The punctate peri-nuclear staining pattern was associated with ER-alpha positivity and was present mainly in well-differentiated samples. In striking contrast, the diffuse staining pattern was present in poorly differentiated samples, and was not associated with ER-status. DNA sequence analysis revealed that only well-differentiated samples with a punctate staining pattern harbored the Cav-1 P132L mutation. Thus, immunostaining of Cav-1 can be used as a first step to stratify human breast patients and to predict the presence of Cav-1 mutations. As such, the punctate Cav-1 immunostaining pattern can now be used as a screening tool to select patients for Cav-1 mutational analysis.     

干扰Sirt2促进C2C12成肌细胞分化

Sirt2是组蛋白去乙酰化酶(HDAC III)家族成员之一, 对细胞周期、自噬、脂肪细胞分化、神经细胞存活等生物学过程的调节发挥重要作用. 目前,Sirt2在肌肉发育过程中的研究尚未见报道.本文通过构建Sirt2慢病毒干扰载体,侵染C2C12成肌细胞,并用细胞免疫荧光化学、real-time PCR 和Western印迹方法,检测其对成肌分化标志基因及相关信号通路因子的影响. 结果显示,干扰质粒shRNA 663处理C2C12细胞后,Sirt2 mRNA及蛋白质表达水平与对照相比显著下调(P<0.01);C2C12细胞分化第4 d,MyoD,MyoG,MyHC mRNA及蛋白质表达均显著增加(P<0.01); PI3K,AKT,FoxO1磷酸化水平明显升高. 结果表明,Sirt2可通过PI3K/AKT/FOXO1信号通路来促进成肌细胞分化,是肌生成的一个潜在调节因子.     

Tyrosine phosphorylation of caveolin-2 at residue 27: differences in the spatial and temporal behavior of phospho-Cav-2 (pY19 and pY27)

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct properties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functional analysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, but clearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstream mediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that in A431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However, the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporal response to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylation at tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to the common characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) may each possess a set of unique functional roles.     

Evidence for Na+/Ca2+ exchanger 1 association with caveolin-1 and -2 in C6 glioma cells

The purpose of this study is to understand the interaction of Na + -Ca2+ exchanger (NCX1), that is one of the essential regulators of Ca2+ homeostasis, with caveolin (Cav)-1 and Cav-2 in Cav-3 null cell (rat C6 glioma cell). Both mRNA and protein expression of NCX1, Cav-1 and Cav-2 was observed, but no expression of mRNA and protein of Cav-3 were observed in C6 glioma cells. In isolated caveolae-enriched membrane fraction, the NCX1, Cav-1 and Cav-2 proteins localized in same fractions. The experiment of immuno-precipitation showed complex formation between the NCX1 and Cavs. Confocal microscopy also supported co-localization of NCX1and Cavs at the plasma membrane. Functionally, sodium-free induced forward mode of NCX1 attenuated by Cav-1 antisense ODN. When treated cells with Cav-2 antisense ODN, both reverse and forward mode of NCX1 was attenuated. From these results, in the Cav-3 lacking cells, the function of NCX1 might be regulated by binding with Cavs. Considering the decrement of NCX1 activity by antisense ODNs, caveolins may play an important role in diverse of pathophysiological process of NCX1-related disorders in the body.     

Regulation of Glycogen Content in Astrocytes via Cav-1/PTEN/AKT/GSK-3β Pathway by Three Anti-bipolar Drugs

Here we present the data indicating that chronic treatment with three antibipolar drugs, lithium, carbamazepine and valproic acid regulates Cav-1/PTEN/PI3K/AKT/GSK-3β signalling pathway and glycogen content in primary cultured astrocytes. All three drugs down-regulate gene expression of Caveoline 1 (Cav-1), decrease membrane content of phosphatase and tensin homolog (PTEN), increase activity of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serine-threonine kinase (AKT), and elevate glycogen synthase kinase 3β (GSK-3β) phosphorylation thus suppressing its activity. As expected, treatment with any of these three drugs increases glycogen content in astrocytes. Our findings indicate that regulation of glycogen content via Cav-1/PTEN/AKT/GSK-3β pathway by the three anti-bipoar drugs may be responsible for therapeutic effects of these drugs, and Cav-1 is an important signal element that may contribute to pathogenesis of various CNS diseases and regulation of its gene expression may be one of the underlying mechanisms of drug action for antibipolar drugs and antidepressants currently in clinical use.     

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions.     

干扰Sema7A抑制C2C12成肌细胞的增殖和分化

为研究脑信号蛋白家族（Semaphorins）成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA（small interfering RNA,siRNA）,用此siRNA转染C2C12成肌细胞．通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,real-time qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高．免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低．Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降．进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调．以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.     

Mechanical stretch activates glycometabolism-related enzymes via estrogen in C2C12 myoblasts

Moderate exercise improves glycometabolic disorder and type 2 diabetes mellitus in menopausal females. So far, the effect of exercise-induced estrogen on muscular glycometabolism is not well defined. The current study was designed to explore the effect of mechanical stretch-induced estrogen on glycometabolism in mouse C₂C₁₂ myoblasts. The mouse C₂C₁₂ myoblasts in vitro were assigned randomly to the control (C), stretch (S), and stretch plus aromatase inhibitor anastrozole (SA) groups. Cells in the S group were stretched by the Flexcell FX-5000™ system (15% magnitude, 1 Hz frequency, and 6-hr duration) whereas those in the SA group were treated with 400 μg/ml anastrozole before the same stretching. Glucose uptake, estradiol levels, PFK-1 levels, and oxygen consumption rate were determined, and the expression of HK, PI3K, p-AKT, AKT, and GLUT4 proteins were semiquantified with western blot analysis. Compared to the control, the estradiol level, oxygen consumption rate, expression of HK, PI3K, and PFK-1 proteins, the ratio of p-AKT to AKT, and the ratio of GLUT4 in the cell membrane to that in the whole cell were higher in the S group. On the other hand, the estradiol level, glucose uptake, expression of PFK-1 and GLUT4 proteins, oxygen consumption rate, expression of HK protein, and the ratio of p-AKT/AKT were lower in the myoblasts in the SA group than those in the S group. The level of estradiol was positively correlated with glucose uptake (p < .01, r = .818). Therefore, mechanical stretch-induced estrogen increased the expression of glycometabolism-related enzymes and proteins in the mouse C₂C₁₂ myoblasts.     

口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达

利用定点突变的原理,获得包含有口蹄疫病毒P1,2A,3C及部分2B编码区的目的基因片段,KpnⅠ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1（+）,经筛选、鉴定及DNA序列分析后,将重组质粒pcDNA3.1/P12X3C转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法,检测细胞中表达的口蹄疫病毒抗原。结果表明,口蹄疫病毒基因片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3.1/P12X3C可在BHK-21细胞中表达FMDV目的蛋白。     

Dexmedetomidine attenuates lipopolysaccharide-induced acute liver injury in rats by inhibiting caveolin-1 downstream signaling pathway

Objective: The aim of the present study is to investigate the anti-injury and anti-inflammatory effects of dexmedetomidine (Dex) in acute liver injury induced by lipopolysaccharide (LPS) in Sprague–Dawley rats and its possible mechanism.Methods: The acute liver injury model of male rats was established by injecting LPS into tail vein. The mean arterial pressure (MAP) of rats was recorded at 0–7 h, and lactic acid was detected at different time points. Wet/dry weight ratio (W/D) was calculated. Pathological changes of rat liver were observed by HE staining. ALT and AST levels in serum were detected. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) in liver tissue homogenate and the levels of IL-1β and IL-18 in serum were detected by ELISA. Protein levels of Caveolin-1 (Cav-1), TLR-4 and NLRP3 in liver tissue were tested by immunohistochemistry method. The expression of Cav-1, TLR-4 and NLRP3 mRNA in liver tissue was detected by quantitative polymerase chain reaction (qPCR) to explore its related mechanism.Results: Compared with NS group, serum lactic acid, W/D of liver tissue, MPO, SOD, IL-1β and IL-18 were significantly increased and MAP decreased significantly in LPS group and D+L group. However, compared with NS group, D group showed no significant difference in various indicators. Compared with LPS group, MPO, SOD, IL-1β and IL-18 were significantly decreased and MAP was significantly increased in D+L group. D+L group could significantly increase the level of Cav-1 protein and decrease the level of TLR-4 and NLRP3 protein in liver tissue caused by sepsis. The expression of Cav-1 mRNA was significantly up-regulated and the expression of TLR-4 and NLRP3 mRNA was inhibited in D+L group.Conclusion: Dex pretreatment protects against LPS-induced actue liver injury via inhibiting the activation of the NLRP3 signaling pathway by up-regulating the expression of Cav-1 by sepsis.     

Point mutated caveolin-3 form (P104L) impairs myoblast differentiation via Akt and p38 signalling reduction, leading to an immature cell signature

Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-β-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-β signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.     

A conserved sequence in caveolin-1 is both necessary and sufficient for caveolin polarity and cell directional migration

Caveolin-1 (Cav-1) plays an important role in the organization of signaling molecules involved in a variety of signaling pathways, including those mediating cell motility. Here we show that amino acids K47-K57 of Cav-1 are a highly conserved sequence in Cav-1 and Cav-3 proteins, and that expression of either K47-K57 deletion Cav-1 mutant or wild-type Cav-2 that lacks this sequence exhibits a non-polarized distribution pattern. Expression of K47-K57 in Cav-2 leads to Cav-2 polarity, suggesting that expression of K47-K57 is sufficient to direct caveolin polarity. Importantly, we show that expression of this sequence is both necessary and sufficient to promote cell directional migration. Thus, our results support the conclusion that Cav-1 polarity is critical for cell directional migration.