Visualization of Actin Filament Patterns in Pollen Tubes of Hosta caerulea Tratt. with a Non-Fixation and TRITC-Phalloidin Method

Actin filament patterns during pollen germination in Hosta caerulea Tratt. were visualized with a simple method in which there was no pre-fixation, with dimethylsulphoxide (DMSO) as a permeabilising agent and staining with TRITC-Phalloidin. The cytoplasm of the vegetative cell of the ungerminated pollen grain contained numerous crystalline fusiform bodies to constitute a storage form of actin. These bodies were transferred to the emerging pollen tube after the germination of the pollen grain. Following the growth of pollen tube, the fusiform bodies were gradually dissociated, branched, slenderized and formed a cross-linked actin network. During the further growth of the pollen tube, the preponderance of longitudinally-oriented thin actin filaments with some anastomoses to form a more complex network present always in the long pollen tube. This was the typical pattern of actin filaments in most cases. In some conditions, actin filaments were assembled to form thick actin cables near the proximate part of the pollen tube tip. The branching and connecting of the cables were probably also seen in some parts. Actin filaments were always entering to the apical region of a tube tip. The significance of the non-fixation and fluorescence-phalloidin (FI-Ph) method and the problems in the future studies are discussed     

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15-90 degrees to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70 degrees in a dendritic array.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.     

用非固定的荧光探针方法观察紫萼花粉管肌动蛋白纤丝的形式

用非固定的、二甲基亚砜作为渗透剂的、异硫氰四甲基罗丹明标记的鬼笔环肽染色方法,观察了紫萼(Hosta caerulea Tratt.)未萌发的花粉粒及不同生长时期的花粉管中的肌动蛋白纤丝的形式。显示未萌发的花粉粒中具有结晶状的梭状体,为肌动蛋白的一种贮藏形式。花粉萌发时,这种梭状体转移到短的花粉管中,逐渐松解、分支和形成肌动蛋白纤丝交错的网络。在花粉管迅速生长和达到一定长度时,肌动蛋白纤丝形成以与花粉管长轴平行的细丝占优势的网络系统,这是在大多数情况中紫萼花粉管典型的肌动蛋白纤丝的形式。在某些条件下,在花粉管接近顶端的前部,肌动蛋白纤丝可集合成长的粗束,这种粗束也常有分支和并合。肌动强白纤丝一直分布到花粉管的末端。讨论了研究肌动蛋白纤丝的非固定方法的重要性和进一步研究花粉管肌动蛋白纤丝值得注意的问题。     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

Fluorescence Microscopic Observations on the Distribution Patterns of Actin Filaments in the Cells of Developing Endosperm in Triticum aestivum

The presence and distribution patterns of actin filaments (AFs) in the cells of developing wheat (Triticum aestivum L. ) endosperm exhibiting intercellular protoplasmic movement were studied with fluorescence microscopy and video microscopy. By using TRITC-PhaIloidin as fluorescence probe and cytochalasin B (CB) treatment it was uncovered that there were a lot of AFs scattered throughout the cytoplasm and the patterns of AFs varied greatly with the actin localization. Four configurations of AFs could be recognized: an actin meshwork surrouding the nucleus; bundles of AFs radiating from nuclear “basket” and extending to the periplasm; numerous finer AFs densely and randomly distributed in the cortical cytoplasm and fusiform bodies composed of AFs appearing in the endosperm cells lying at the “cheek” of the caryopsis. Judging from the dynamic characters of intercellular movement of the cytoplasmic constituents and the reaction of cytoplasmic strands related to CB treatment, the authors have discussed and proposed that the exhibition of the two kinds of intercellular movement (extrusion of cytoplasmic strands and mass flow of ground substance) might also be in close relation to the different configurations of AF organization in the cytoplasm.     

The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages

A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin- binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maintenance of the intestinal tube in Caenorhabditis elegans: the role of the intermediate filament protein IFC-2

The Caenorhabditis elegans intestinal lumen is surrounded by a dense cytoplasmic network that is laterally attached to the junctional complex and is referred to as the endotube. It localizes to the terminal web region which anchors the microvillar actin filament bundles and is particularly rich in intermediate filaments. To examine their role in intestinal morphogenesis and function, C. elegans reporter strains were generated expressing intestine-specific CFP-tagged intermediate filament polypeptide IFB-2. When these animals were treated with dsRNA against intestinal intermediate filament polypeptide IFC-2, the endotube developed multiple bubble-shaped invaginations that protruded into the enterocytic cytoplasm. The irregularly widened lumen remained surrounded by a continuous IFB-2::CFP-labeled layer. Comparable but somewhat mitigated phenotypic changes were also noted in wild-type N2 worms treated with ifc-2 (RNAi). Junctional complexes were ultrastructurally and functionally normal and the apical domain of intestinal cells was also not altered. These observations demonstrate that IFC-2 is important for structural maintenance of the intestinal tube but is not needed for establishment of the endotube and epithelial cell polarity.     

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.     

Characterization of intermediate filaments and their structural organization during epithelium formation in pigmented epithelial cells of the retina in vitro

Summary Retinal pigmented epithelial cells of chicken have circumferential microfilament bundles (CMBs) at the zonula adherens region. Isolated CMBs are polygons filled with a meshwork composed primarily of intermediate filaments; they show three major components of 200000, 55000, and 42000 daltons in SDS-gel electrophoresis. Here we have characterized the 55000-dalton protein immunochemically and ultrastructurally. Immunoblotting and immunofluorescence microscopy have shown that the 55000-dalton protein is an intermediate filament protein, vimentin.Vimentin filaments changed their distribution during differentiation of pigmented epithelial cells in culture. The protein in the elongated cells showed a fibroblast-type pattern of intermediate filaments. During epithelium formation, the filaments were uniformly distributed and formed a finer meshwork at the apical level. In pigmented epithelial cells that differentiated and matured in culture, vimentin and actin exhibited their characteristic behavior after treatment with colcemid. In the central to basal region of the cell, intermediate filaments formed thick perinuclear bundles. In the apical region, however, intermediate filaments changed in organization from a nonpolarized meshwork to a polarized bundle-like structure. Simultaneously, new actin bundles were formed, running parallel to the intermediate filaments. This suggests that there is some interaction between microfilaments and intermediate filaments in the apical region of these cells.     

Leading edge movement and ultrastructure in mouse macrophages

The first event in the process of translocation of a cell over a substrate is the forward protrusion of a thin layer of cytoplasm, sometimes referred to as the leading edge. To gain more direct information on structural reorganizations associated with protrusion we have documented the ultrastructure of the actin cytoskeleton of mouse macrophages whose history of locomotion prior to fixation for electron microscopy had been recorded by video microscopy. It is shown that rapid protrusion is associated with the formation of a dense, diagonal network of actin filaments, lacking organized bundles. In cell edges that showed minor fluctuations back and forth over a period of 30 sec or more no dense meshworks were found: instead, a loose peripheral bundle of actin filaments was commonly observed. Cell edges that first protruded and then retracted showed a similar ultrastructure to those that exhibited only forward movement, but the width of the leading edge meshwork was, by comparison, reduced. Measurements showed that there was an approximate correlation between the leading edge mesh width and the net forward translocation observed during the terminal 30 sec, up to fixation. The results are discussed in relation to present concepts of the protrusion mechanism.     

Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum

We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.     

Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.     

Modulation of cytokeratin and actin gene expression and fibrillar organization in cultured rat hepatocytes.

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.     

Filopodial initiation and a novel filament-organizing center, the focal ring

This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure approximately 120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) "axial" filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) "lateral" filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.     

Golgi-associated filament networks in duct epithelial cells of rabbit submandibular glands: immunohistochemical light and electron microscopic studies

The duct epithelial cells of rabbit submandibular glands expressed keratin 8, keratin 14, and actin in the supranuclear region, and these cytoskeletal proteins formed ring structures, approximately 1-2.5 microm in diameter. Ultrastructurally, these ring structures were observed as a 'Golgi-associated filament network' surrounding Golgi apparatuses. Double immunofluorescent studies showed that keratin 8 and keratin 14 formed keratin 8/14 filaments, and that these filaments and actin filaments colocalized as components of the Golgi-associated filament network. Our studies suggested that the Golgi-associated filament network maintains the complex structure and location of the Golgi apparatus of the duct epithelium of rabbit submandibular glands. Tubulin was distributed diffusely throughout the cytoplasm of columnar cells, but no special relationship was found between tubulin and the Golgi apparatus.     

Actin filament array during side branch initiation in protonema cells of the mossFunaria hygrometrica: an actin organizing center at the plasma membrane

Summary With an improved method to visualize the actin filament system it is possible to detect a small, peculiar accumulation of actin filaments under the prospective area of side branch formation inFunaria protonema cells. It consists of a ring-like configuration of actin filaments from which filaments radiate, preferentially along the plasma membrane. During the transition to tip growth the arrangement becomes loosened and eventually disappears whereas the filaments are concentrated in inner regions of the cytoplasm with a maximum in the apical dome.