Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK-I and ROCK-II

Rho-associated coiled-coil-forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK-I and ROCK-II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross-mating of ROCK-I(+/-) ROCK-II(+/-) double heterozygous mice showed that all of the ROCK-I(-/-) ROCK-II(-/-) homozygous mice die in utero before 9.5 days post-coitum (dpc) and ROCK-I(-/-) ROCK-II(+/-) homo-heterozygous or ROCK-I(+/-) ROCK-II(-/-) hetero-homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK-I(+/-) ROCK-II(-/-) or ROCK-I(-/-) ROCK-II(+/-) mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild-type embryos treated ex vivo with a ROCK inhibitor, Y-27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.     

MicroRNAs control eyelid development through regulating Wnt signaling

Background: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. Results: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer ^fl/fl;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer ^fl/fl;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial–mesenchymal interactions in eyelid development. Conclusions: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.     

The unfolded protein response in familial amyotrophic lateral sclerosis

Mutant superoxide dismutase type 1 (MTSOD1) is thought to cause ～20% of cases of familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Previous studies have shown that MTSOD1 accumulates inside the endoplasmic reticulum (ER) and activates the unfolded protein response (UPR), suggesting that ER stress is involved in the pathogenesis of FALS. We used a genetic approach to investigate the role of the UPR in FALS. We crossed G85RSOD1 transgenic mice with pancreatic ER kinase haploinsufficient (PERK(+/-)) mice to obtain G85R/PERK(+/-) mice. PERK(+/-) mice carry a loss of function mutation of PERK, which is the most rapidly activated UPR pathway, but have no abnormal phenotype. Compared with G85R transgenic mice, G85R/PERK(+/-) mice had a dramatically accelerated disease onset as well as shortened disease duration and lifespan. There was also acceleration of the pathology and earlier MTSOD1 aggregation. A diminished PERK response accelerated disease and pathology in G85R transgenic mice presumably because the mice had a reduced capacity to turn down synthesis of misfolded SOD1, leading to an early overloading of the UPR. The results indicate that the UPR has a significant influence on FALS, and suggest that enhancing the UPR may be effective in treating ALS.     

Smad4对小鼠眼睑发育的影响

目的: 利用眼特异性 Smad4 基因敲除小鼠,观察其眼睑表型并探讨其眼睑发育异常的可能机制。方法: 选择 PAX6 第一启动子P0驱动的晶体外胚层特异性表达 Cre重组酶的转基因小鼠(Le-Cre)作为介导敲除的工具鼠,将其与 Smad4 条件基因小鼠( Smad4 ^fl/fl)交配获得 Le-Cre特异性Smad4 基因敲除小鼠,通过HE染色揭示其眼睑组织形态学的改变,采用免疫染色技术对某些关键蛋白的表达进行检测并与野生型小鼠进行比较,并检测其眼睑上皮细胞凋亡和增殖的改变。结果: Smad4 在眼睑的失活导致眼睑在发育过程中不能融合,生后眼睑保持开放;Smad4在眼睑的表达缺失不影响眼睑睑缘上皮细胞的增殖和凋亡,但导致上皮细胞内c-Jun磷酸化过程受损,表皮生长因子受体(EGFR)核转位受影响,引起细胞内肌动蛋白束装配异常而导致上皮细胞移行受损,出现眼睑发育时融合不能。结论: Smad4在眼睑发育中对于眼睑的闭合是必需的。     

氯化锂对FMR1基因敲除小鼠的高架十字迷宫行为的干预作用

目的 探讨糖原合成酶激酶3（GSK3）抑制剂氯化锂对FMR1基因敲除（KO）小鼠的高架十字迷宫行为的干预作用及机制。方法 给90只30日龄FMR1基因敲除小鼠连续5d腹腔注射不同剂量的氯化锂，用药第6天进行高架十字迷宫行为学实验，通过录像机录像，然后用Smart软件分析录像，观察能否改善KO鼠的高架十字迷宫的表型；同时通过免疫印迹技术检测KO及野生型（WT）鼠的海马和皮层中GSK3β和磷酸化GSK3β（p-GSK3β）的变化。结果 在高架十字迷宫实验中，与WT组小鼠比较，KO组小鼠的运动性、兴奋性、探索性明显增强，且KO组小鼠在开放区域的活动时间、次数以及路程均明显高于WT组。KO鼠用氯化锂后，在开放区域的活动时间、次数以及路程均减低，差异具有统计学意义（P ＜0.05）。免疫印迹实验结果显示，KO鼠p GSK3β表达比WT鼠少；用氯化锂后，KO鼠p-GSK3β表达增加。WT鼠用氯化锂后，开放区域活动时间、次数以及路程有较少改善，p-GSK3β表达也有增加。未使用氯化锂的KO鼠与WT鼠相比，总GSK3β表达无明显差异，KO鼠和WT鼠使用氯化锂后，总GSK3β无明显改变，P ＞0.05。结论 GSK3β的抑制剂氯化锂能改善KO鼠的高架十字迷宫表型，对KO鼠有治疗作用，其机制可能与氯化锂导致的p-GSK3β表达增加有关。     

Effects of in vivo heregulin beta1 treatment in wild-type and ErbB gene-targeted mice depend on receptor levels and pregnancy

Mice heterozygous (+/-) for either heregulin (HRG), ErbB2, or ErbB3 were created by gene targeting, resulting in the loss of one functional gene copy and an associated decrease in targeted protein. We examined the in vivo activity of recombinant HRG peptide, rHRG beta1 (amino acids 177 to 241), in the three heterozygous mouse lines and in wild-type (WT) mice, both pregnant and nonpregnant. Nonpregnant WT and HRG(+/-) mice of both sexes were sensitive to rHRG beta1 treatment as evidenced by a high mortality rate associated with abdominal enlargement and parietal cell loss. However, pregnant WT mice and ErbB2 and ErbB3 heterozygous mice treated with rHRG beta1 were less affected, with significantly lower mortality rates and a less severe abdominal phenotype. Histological analysis revealed extensive breast ductal hyperplasia in females of all genotypes after rHRG beta1 treatment. Hyperplasia of other epithelial tissues such as the pancreas and intestine and the growth of cardiac nerve bundles were also observed, independent of sex.     

ROCK-II mediates colon cancer invasion via regulation of MMP-2 and MMP-13 at the site of invadopodia as revealed by multiphoton imaging

The ROCK-II isoform of Rho's downstream effector, Rho kinase, has been linked with greater invasion and metastasis in solid tumors. We have previously shown that ROCK-II is overexpressed at the advancing edge of colon cancers. The mechanism whereby ROCK-II contributes invasion, particularly in the setting of colon cancer, remains to be elucidated fully. To better understand its contribution, we evaluated ROCK-II expression in both non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines grown in type I collagen scaffolds. Using multiphoton microscopy, we observed that ROCK-II localized to the actin cytoskeleton in non-malignant cells but localized to the cell periphery as focal collections with an absence of adjacent collagen in all colon cancer cell lines. By transmission electron microscopy, these collections corresponded with finger-like projections previously described as invadopodia. Immunogold staining with cortactin, matrix metalloprotease (MMP)-2, -9, and -13 confirmed that these were indeed invadopodia. To further link ROCK-II to colon cancer invasion, we treated non-malignant and malignant intestinal epithelial cell lines with ROCK-II siRNA and evaluated depth of invasion, proliferation, and MMP-2, -9, and -13 activities. The most striking effect was seen in the highly tumorigenic cell lines, SW620 and HCT-116, wherein ROCK-II knockdown resulted in a two-fold or more reduction in invasion. This reduction in invasion was not due to a decrease in cell proliferation, as a significant reduction in proliferation was only observed in the two non-malignant intestinal cell lines. Finally, both MMP-2 and -13 activities were significantly decreased in all colon cancer cell lines. Taken together, these data suggest for the first time that ROCK-II is a critical mediator of colon cancer cell invasion through its modulation of MMP-2 and -13 at the site of invadopodia but regulates proliferation in non-malignant intestinal cells.     

Breathing dysfunctions associated with impaired control of postinspiratory activity in Mecp2−/y knockout mice

Rett syndrome (RTT) is an inborn neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 gene ( MECP2 ). Besides mental retardation, most patients suffer from potentially life-threatening breathing arrhythmia. To study its pathophysiology, we performed comparative analyses of the breathing phenotype of Mecp2^−/y knockout (KO) and C57BL/6J wild-type mice using the perfused working heart–brainstem preparation (WHBP). We simultaneously recorded phrenic and efferent vagal nerve activities to analyse the motor pattern of respiration, discriminating between inspiration, postinspiration and late expiration. Our results revealed respiratory disturbances in KO preparations that were similar to those reported from in vivo measurements in KO mice and also to those seen in RTT patients. The main finding was a highly variable postinspiratory activity in KO mice that correlated closely with breathing arrhythmias leading to repetitive apnoeas even under undisturbed control conditions. Analysis of the pontine and peripheral sensory regulation of postinspiratory activity in KO preparations revealed: (i) prolonged apnoeas associated with enhanced postinspiratory activity after glutamate-induced activation of the pontine Kölliker-Fuse nucleus; and (ii) prolonged apnoeas and lack of reflex desensitization in response to repetitive vagal stimulations. We conclude that impaired network and sensory mediated synaptic control of postinspiration induces severe breathing dysfunctions in Mecp2^−/y KO preparations. As postinspiration is particularly important for the control of laryngeal adductors, the finding might explain the upper airway-related clinical problems of patients with RTT such as apnoeas, loss of speech and weak coordination of breathing and swallowing.     

Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing.     

DSTYK kinase domain ablation impaired the mice capabilities of learning and memory in water maze test

DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.     

Tyrosine kinase activity and remodelling of the actin cytoskeleton are co-temporally required for degranulation by cytotoxic T lymphocytes

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation. This signal reversal is not restricted to tyrosine phosphorylation, as disruption of the actin cytoskeleton also reverses the phosphorylation of the more downstream serine/threonine kinase extracellular signal regulated kinase (Erk). An intact cytoskeleton and cell spreading are not sufficient for maintaining signals, as stabilization of actin filaments, at a point when peak tyrosine phosphorylation is occurring, also leads to the rapid loss of protein tyrosine phosphorylation. Disruption of tyrosine kinase activity after TCR signals are maximally induced causes the immediate reversal of tyrosine phosphorylation as well as cytoskeletal disruption, as indicated by loss of cell spreading, adhesion and CTL degranulation. Taken together, our results indicate that actin remodelling occurs co-temporally with ongoing tyrosine kinase activity, leading to CTL degranulation. We hypothesize that continuous actin remodelling is important for sustaining productive signals, even after downstream signalling molecules such as Erk have been activated, and that the actin cytoskeleton is not solely required for initiating and maintaining the T cell in contact with its stimulus.     

Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells

Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.     

N-ethyl-N-nitrosourea (ENU)-induced meningiomatosis and meningioma in p16(INK4a)/p19(ARF) tumor suppressor gene-deficient mice

The cyclin-dependent kinase (CDK) inhibitor p16(INK4a) and the MDM2 ubiquitin ligase inhibitor p19(ARF) are critical to the regulation of cell cycle progression. Their loss by deletion, mutation or epigenetic silencing is a common molecular alteration in many human cancers. To investigate the role of p16(INK4a)/p19(ARF) deficiency in CNS tumor pathogenesis, pregnant mice bearing p16(-/-)/p19(-/-), p16(+/-)/p19(+/-), and p16(+/+)/p19(+/+) embryos were exposed transplacentally on gestation day 14 to a single dose of the potent carcinogen, ethylnitrosourea (ENU). p16(+/-)/p19(+/-) male mice treated with ENU developed meningial proliferative lesions with a high incidence (5/10). The incidence was lower in other ENU-treated animals of both sexes and none occurred in saline-treated control animals. The lesions ranged from widespread meningeal proliferation and plaque-like thickening by neoplastic spindle cells consistent with meningiomatosis to a larger discrete mass consistent with a meningioma. Ultrastructural analysis revealed the presence of intercellular junctions between cells, supporting a meningothelial histogenesis. Spontaneous meningiomas occur rarely in wild-type mice but are a common neoplasm afflicting humans, accounting for between 13 and 26% of primary intracranial neoplasms. This ENU inducible meningeal lesion in p16(+/-)/p19(+/-) mice may provide additional insight into the pathogenesis of meningeal neoplasia and aid the development of therapeutics.     

Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart

Summary The role of actin bundles on the heart looping of chick embryos was examined by using cytochalasin B, which binds to the barbed end of actin filaments and inhibits association of the subunits. It was applied to embryos cultured according to New's method. Looping did not occur when cytochalasin B was applied diffusely in the medium. Further, we disorganized actin bundles in a limited part of the heart tube to examine the role of actin bundles in each part in asymmetry formation. A small crystal of cytochalasin B was applied to the caudal part of the heart tube on either the left or right side. The disorganization of actin bundles on the left side resulted in the right-bending of the heart, an initial sign of dextro-looping (normal pattern), and right side disorganization resulted in left-bending. We suggest that actin bundles on the right side of the caudal part of a heart tube generate tension and cause dextro-looping. Embryos whose hearts bent to the right rotated their heads to the right, and embryos with left-bent-hearts rotated their heads to the left. The rotation of the heart tube may therefore decide in which direction the body axis rotates.     

A mouse model for hereditary hemorrhagic telangiectasia (HHT) type 2

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal-dominant disorder characterized by the age-dependent development of focal arteriovenous malformations and telangiectases. HHT type 2 is caused by loss of function mutations in activin receptor-like kinase 1 (ACVRL1 or ALK1). However, the factors that initiate lesion formation and those that influence disease progression remain unknown. Because heterozygous mice contain the appropriate genotype for an animal model of this disorder, mice heterozygous for a loss-of-function mutation in Acvrl1 were carefully examined for an HHT-like phenotype. These mice developed age-dependent vascular lesions in the skin, extremities, oral cavity and in the internal organs (lung, liver, intestine, spleen and brain), similar to those seen in HHT patients. Major histopathological features of the lesions included thin-walled dilated vessels in close proximity to each other, hemorrhage and fibrosis. Similar to HHT patients, the mice also exhibited gastrointestinal bleeding, as evidenced by positive fecal occult blood tests. An Acvrl1(+/-) mouse with profound liver involvement also displayed a secondary cardiac phenotype, similar to that observed in human patients. The similarity of affected organs, age-dependent penetrance, histological similarity of the lesions and recapitulation of a secondary phenotype suggest that the Acvrl1(+/-) mice are an appropriate animal model for the identification of additional genetic and environmental factors that cause pathology in HHT type 2 patients. In addition, studies utilizing this animal model can yield valuable information on the role of ALK1 in maintenance of adult vascular architecture including arteriovenous identity.     

Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing

Animal cornea is an avascular transparent tissue that is suitable for research on wound healing-related scarring and neovascularization. Here we show that loss of tumor necrosis factor alpha (TNFalpha) potentiates the undesirable, pathogenic response of wound healing in an alkali-burned cornea in mice. Excessive invasion of macrophages and subsequent formation of a vascularized scar tissue were much more marked in TNFalpha-null knockout (KO) mice than in wild-type mice. Such an unfavorable outcome in KO mice was abolished by Smad7 gene introduction, indicating the involvement of transforming growth factor beta or activin/Smad signaling. Bone marrow transplantation from wild-type mice normalized healing of the KO mice, suggesting the involvement of bone marrow-derived inflammatory cells in this phenomenon. Co-culture experiments showed that loss of TNFalpha in macrophages, but not in fibroblasts, augmented the fibroblast activation as determined by detection of alpha-smooth muscle actin, the hallmark of myofibroblast generation, mRNA expression of collagen Ialpha2 and connective tissue growth factor, and detection of collagen protein. TNFalpha in macrophages may be required to suppress undesirable excessive inflammation and scarring, both of which are promoted by transforming growth factor beta, and for restoration of tissue architecture in a healing alkali-burned cornea in mice.     

Mice deficient for cytosolic thymidine kinase gene develop fatal kidney disease

The thymidine kinase (Tk) gene codes for a cytosolic protein involved in the pyrimidine nucleotide salvage pathway. A functional Tk gene is not necessary for cells in culture, and a naturally occurring Tk deficient phenotype has not been described in humans or animal models. In order to determine the biological significance of the Tk gene, we created Tk(-/-) knockout (KO) mice through homologous recombination in mouse embryonic stem cells. Tk KO mice have shortened life spans compared with their wild-type or Tk heterozygous (HET) siblings. All Tk KO mice develop sclerosis of kidney glomeruli and die before one year of age of kidney failure. Among other changes in KO animals, the most consistent is a switch from exclusively mucous secretion to predominantly serous secretion in the sublingual salivary gland. HET parents can produce KO mice at a frequency approaching Mendelian inheritance. Other observations in KO animals include an elevated level of serum thymidine, a significant decrease in the cloning efficiency of splenic lymphocytes, an increase in the frequency of hypoxanthine guanine phosphoribosyl transferase gene mutant lymphocytes, and histological alteration in the lymphoid structure of the spleen. In addition, KO animals sporadically exhibit inflammation of the arteries, which taken together with the lymphocyte and spleen abnormalities, suggest an abnormal immune system. Alterations in Tk KO mice indicate that the pyrimidine nucleotide salvage pathway is indispensable in vivo.     

Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.     

Focal adhesion kinase controls pH-dependent epidermal barrier homeostasis by regulating actin-directed Na+/H+ exchanger 1 plasma membrane localization

Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.     

Actin filament organization in aligned prefusion myoblasts

The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.