Actin Cytoskeleton ; Electromagnetic Fields

Filamin A is an actin-binding protein and, in humans, is encoded by FLNA gene in the long arm of X chromosome. Filamin A plays a role in the formation of cytoskeleton by crosslinking actin filaments in cytoplasm. FLNA mutations affect cytoskeletal regulatory processes and cellular migrating abnormalities that result in periventricular heterotopia. A 5-month-old girl was hospitalized because of breathing difficulty and was diagnosed as having periventricular heterotopia with laryngomalacia, cricopharyngeal incoordination, pulmonary hypertension, and chronic lung disease. A genetic test was performed to find the cause of periventricular heterotopia, and FLNA gene mutation (c.5998+1G>A) was confirmed for the first time in Korea. After discharge, she developed respiratory failure due to a viral infection at 8 months of her age. In spite of management with mechanical ventilation, she died of pneumothorax and pulmonary hemorrhage. Herein, we report a case of FLNA gene mutation who presented with periventricular nodular heterotopia with respiratory insufficiency.
Actin Cytoskeleton ; Arm ; Ataxia ; Cytoplasm ; Cytoskeleton ; Female ; Filamins ; Hemorrhage ; Humans ; Hypertension, Pulmonary ; Infant ; Korea ; Laryngomalacia ; Lung Diseases ; Periventricular Nodular Heterotopia ; Pneumothorax ; Respiration ; Respiration, Artificial ; Respiratory Insufficiency ; X Chromosome

PURPOSE: In osteoclast differentiation, actin-rich membrane protrusions play a crucial role in cell adhesion. Odontogenic ameloblast-associated protein (Odam) contributes to cell adhesion by inducing actin rearrangement. Odam-mediated RhoA activity may play a significant role in multinucleation of osteoclasts. However, the precise function of Odam in osteoclast cell adhesion and differentiation remains largely unknown. Here, we identify a critical role for Odam in inducing osteoclast adhesion and differentiation. MATERIALS AND METHODS: The expression of Odam in osteoclasts was evaluated by immunohistochemistry. Primary mouse bone marrow and RAW264.7 cells were used to test the cell adhesion and actin ring formation induced by Odam. RESULT: Odam was expressed in osteoclasts around alveolar bone. Odam transfection induced actin filament rearrangement and cell adhesion compared with the control or collagen groups. Overexpression of Odam promoted actin stress fiber remodeling and cell adhesion, resulting in increased osteoclast fusion. CONCLUSION: These results suggest that Odam expression in primary mouse osteoclasts and RAW264.7 cells promotes their adhesion, resulting in the induction of osteoclast differentiation.
Actin Cytoskeleton ; Actins* ; Animals ; Bone Marrow ; Cell Adhesion ; Collagen ; Immunohistochemistry ; Membranes ; Mice ; Osteoclasts* ; Stress Fibers ; Transfection

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.
Actin Cytoskeleton ; Cell Body ; Cytoskeleton ; Microscopy, Atomic Force ; Muscle Cells ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Polymerization ; Polymers ; Stem Cells* ; Stress Fibers ; Tissue Engineering

Rho small GTPases control multiple aspects of neuronal morphogenesis by regulating the assembly and organization of the actin cytoskeleton. Although they are negatively regulated by GTPase activating proteins (GAPs), the roles of RhoGAPs in the nervous system have not been fully investigated. Here we describe a characterization of Drosophila RhoGAP68F that is mainly expressed in the embryonic central nervous system. RNA in situ hybridization analysis showed that expression of RhoGAP68F is highly restricted to the embryonic brain and ventral nerve cord. Database search revealed that RhoGAP68F contains an N-terminal Sec14 domain and a C-terminal RhoGAP domain. Rho-GTP pull-down assay demonstrated that the RhoGAP domain of RhoGAP68F inactivates RhoA but not Rac1 or Cdc42 in HEK293 cells. In addition, expression of RhoGAP68F in NIH3T3 cells suppressed LPA-induced stress fiber formation, which is mediated by RhoA. Finally, neuronal overexpression of RhoGAP68F causes synaptic overgrowth at the larval neuromuscular junction (NMJ). Taken together, these results suggest that RhoGAP68F may play a role in synaptic growth regulation by inactivating RhoA.
Actin Cytoskeleton ; Actins ; Brain ; Central Nervous System ; Drosophila ; GTPase-Activating Proteins ; HEK293 Cells ; In Situ Hybridization ; Monomeric GTP-Binding Proteins ; Morphogenesis ; Nervous System ; Neuromuscular Junction ; Neurons ; RNA ; Stress Fibers

Seven fibrovascular diabetic preretinal membranes were examined with lightmicroscophic immunohistochemical stain and electron-microscopy to evaluate the possibility of pericytes to be involved in membrane contraction. Pericytes were positively stained with anti-actin antibody together with some stromal cells thought to be myofibroblasts presumedly. On transmission electron microscopic study, pericytes were highly active with numerous cytoplasmic processes and contained abundant microfilaments considered as actin in their cytoplasm. Pericyte/endothelial cell ratio of vascular channels were increased in some actively proliferative portion of the membranes. Myofibroblasts that contain abundant cytoplasmic microfilaments were also demonstrated in the extravascular stroma of the membranes and were very similar to the pericytes morphologically. Although the evidence that the pericytes are related to the origin of the myofibroblasts could not be demonstrated, this study suggested that the pericytes may play important roles in development and contraction mechanism of fibrovascular diabetic preretinal membranes.
Actin Cytoskeleton ; Actins ; Cytoplasm ; Membranes* ; Myofibroblasts ; Pericytes* ; Stromal Cells

OBJECTIVETo explore the effects of cofilin on the actin cytoskeleton reorganization in osteoblasts induced by fluid shear stress.

METHODSFluid shear stress (1.2 Pa) was applied to osteoblasts for 0 (control group), 15, 30, 45, 60, 120 min in vitro. Cells were stained with fluorescein isothiocyanate (FITC)-phalloidin for fiber-actin, and confocal laser scanning microscope(CLSM) was used to observe the fluorescence of fiber-actin. Western blotting was used to detect the expression of the cofilin and the phospho-cofilin.

RESULTSActin filaments became organized into stress fibers that were thicker and more abundant than those in non-flowed cells. The fluorescence intensity (38.00 ± 6.88) of fiber-actin after 120 min (42.93 ± 6.41) loading it was 2.8 times as much as that in control group (15.41 ± 3.60, P < 0.05). Additionally, the level of phospho-cofilin protein was dramatically elevated after loading. Fluid shear stress induced an initial decrease of cofilin at 60 min. However, at 120 min cofilin (0.254 ± 0.026) increased to 1.5 times as much as that at 60 min (0.162 ± 0.004).

CONCLUSIONSThe results indicate that cofilin phosphorylation mediates fiber-actin reorganization in the osteoblasts induced by fluid shear stress.

The Kingdom fungus has a unique structure and organization. Recent advances in electron microscopy and use of specific cytochemical technique enable the ultrastructures to be visualized. The hypha is a tube-like structure with a rigid wall, containing a moving slug of protoplasm. Hypha grows only at the tapered apical tip region, which is called extension zone. Extreme tip area has apical vesicle cluster which is responsible for tip growth. Unique fungal structure, Spitzenk rper, is thought to be a central region of the apical vesicle cluster. Most hyphal structures except the species belong to Zygomycetes have septa. But the septum is not completely blocked and it has different types of opening pores. The simple septal pores with Woronin bodies, which are found in Ascomycetes and Deuteromycetes, can be plugged in two different mechanisms. During normal differentiation the pores become occluded by a gradual deposition of plugged material. Loss of cytoplasm from damaged hyphae can be reduced and blocked by the rapid occlusion of septal pores by Woronin bodies or hexagonal crystal bodies. Septal sealing in Basidiomycetes which have dolipore septum is made by the rapid formation of electron-dense pore plugs. The shape of the fungal cell is the shape of fungal wall. Fungal walls appear to be composed of layers, which are thought to merge into one another to form one structure. The cytoskeleton consists of microtubules and microfilaments with motor proteins, and they seems to act together in the fungal cells.
Actin Cytoskeleton ; Ascomycota ; Basidiomycota ; Cytoplasm ; Cytoskeleton ; Fungal Structures ; Fungi ; Gastropoda ; Hyphae ; Microscopy, Electron ; Microtubules ; Mitosporic Fungi

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*