Distribution Of Microfilament Bundles Research Articles

Caldesmon is necessary for maintaining the actin and intermediate filaments in cultured bladder smooth muscle cells

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.

Transverse pattern of microfilament bundles induced in epitheliocytes by cylindrical substrata

Cylindrical culture substrata are known to induced longitudinal orientation of polarized fibroblasts and corresponding alignment of actin microfilament bundles in these cells. We studied microfilament bundle distribution in two cell types, fibroblasts and epitheliocytes, spread on two kinds of anisotropic substrata, quartz glass cylinders with a diameter 32 microns and narrow (25-40 microns wide) flat glass adhesive strips with non-adhesive borders. Rat embryo and human diploid fibroblasts, as expected, formed predominantly longitudinally aligned bundles on both substrata. In contrast, transverse bundles on cylinders and randomly oriented bundles on flat strips were formed in IAR-2 and MDCK epithelial cells. We interpret these data as showing that the epitheliocyte attempts to override the guiding influence of anisotropic substrata. The microfilament bundle pattern on cylinders depends on the integrity of the microtubules. Colcemid-induced microtubule depolymerization caused formation of longitudinal as well as transverse bundles both in fibroblasts and epitheliocytes, thus diminishing the differences in microfilament bundle patterns in two cell types. These results show that microtubules control the cell-type-specific distribution of microfilament bundles both in polarized fibroblasts and in discoid epitheliocytes. However, the results of this control are opposite: microtubules enhance cell polarization in fibroblasts, but prevent it in epithelial cells.

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ.

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.

Microfilament bundles in the roots of a conifer,Chamaecyparis obtusa

The distribution of microfilament bundles (MFBs) in the primary tissues ofChamaecyparis obtusa roots has been investigated by electron microscopy. Nomarski differential interference-contrast (NDIC) images of MFBs in sections of embedded materials are also presented to complement the ultrastructural observations. The peripheral phloem parenchyma cells, also known as precursory phloem, generally possess greater numbers of MFBs than do any other cell type. MFBs are apparently absent in the cortical, meristematic or root cap tissues. The number of MFBs seen in a transection of a cell varies according to its position in the ontogenetic sequence. While all the MFBs in peripheral phloem parenchyma cells lie within 2.0 μm from and on occasion contact the plasmamembrane, some MFBs in other phloem and xylem cells are located in the central areas of the cytoplasm. The possible three-dimensional distribution of MFBs in a streaming peripheral phlowm parenchyma cell is discussed.

Microfilaments in plant vascular cells

Microfilaments 50–70 Å (Å = 0.1 nm) in diameter are commonly found in vascular tissue of elongating roots and stems of many plants. Such microfilaments occur in bundles in peripheral regions of elongating or differentiating vascular cells, and are usually oriented parallel to the longitudinal axis of the cells. Although the longest microfilament bundle we have measured is about 15 μm, we suspect that the bundles are at least as long as the cells that contain them. The bundles are 0.1–0.4 μm in width and are often in apparent contact with organelles, but there is no definite indication of their being anchored to plasmalemma or any organelle. The microfilaments are comparable in size and morphology to actin filaments. The distribution of microfilament bundles in the vascular tissue of various plants and the possible function of the bundles is discussed.

Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts

The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.