The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.     

Program of F-actin in the follicular epithelium during oogenesis of the german cockroach, Blattella germanica

Extensive programmed structural and functional changes of insect follicular epithelium during oogenesis provide a model to study modulation of cytoskeletal organization during morphogenesis in a non-dividing cell population. Rhodamine-phalloidin staining of whole mounted and cryosectioned oogenic follicles reveal changing F-actin filament organization from pre- to post-vitellogenic stages consistent with the presumptive dorsal-ventral orientation of the future embryo. Filaments are not abundant in pre-vitellogenic follicle cells up to day 2. Differences between dorsal and ventral follicle cells appear first on day 3. Obviously patent follicle cells are seen only on the ventral follicle surface which exhibits stronger F-actin fluorescence than the dorsal non-patent epithelium. On the presumptive ventral side of midvitellogenic follicles morphologically distinct bundles of actin filaments orient peripherally into projections connecting adjacent follicle cells and from the center of follicle cells apically into macrovillar projections extending toward the oocyte surface. The mid-vitellogenic dorsal follicle cell layer also possesses macrovillar extensions containing F-actin which reach and appear to penetrate the oolema. During chorion deposition major reorganization of actin of follicle cells takes place. After chorion deposition all F-actin filaments within a given follicle cell are arranged into large parallel bundles with semi-regular cross-striations which exclude fluorescent label. The parallel orientation of actin striated filament bundles within each follicle cell appears to be random with respect to the orientation of bundles in neighboring follicle cells over much of the mid-latitude of the follicle epithelium. At anterior and posterior follicle poles a more axial orientation of striated bundles is evident. This muscle-like tissue arrangement is appropriate for cooperation in ovulating the chorionated oocyte from the follicle into the oviduct.     

Colocalization of cortical microtubules and F-actin in<Emphasis Type="Italic">Dipodascus magnusii</Emphasis> using confocal laser scanning microscopy

Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.     

Ultrastructure and function of nurse cells in phthirapterans. Possible function of ramified nurse cell nuclei in the cytoplasm transfer

The structure of nurse cells as well as the distribution of cytoskeletal elements (actin filaments, microtubules) in three representatives of phthirapterans: the pig louse, Haematopinus suis (Anoplura) and bird lice, Eomenacanthus stramineus, Columbicola columbae (Mallophaga) were investigated. All three species have polytrophic-meroistic ovaries which means that each oocyte remains connected with a group of nurse cells via specialized cytoplasmic canals-intercellular bridges (ring canals). Throughout vitellogenesis, various macromolecules as well as organelles (mitochondria, endoplasmic reticulum vesicles, ribosomes) are transferred from the nurse cells to the oocyte. During this flow, the nurse cell nuclei do not enter the oocyte and are retained in the cell centers. In holometabolous insects (e.g. Drosophila, hymenopterans), the central position of nurse cell nuclei is maintained by cytoskeletal elements (actin filaments or microtubules). In the investigated species, the nurse cells are equipped with large, highly extended (irregularly lobed) nuclei. The inner nuclear membrane is lined with a relatively thick layer of nuclear lamina. Ultrastructural analysis and staining with rhodamine-labeled phalloidin revealed that the nurse cell cytoskeleton is poorly developed and represented only by: (1) single microtubules in the perinuclear cytoplasm; and (2) the F-actin layer in the cortical cytoplasm. In the light of this, we postulate that in phthirapterans the position of nurse cell nuclei during the cytoplasm transfer is maintained not by the cytoskeletal elements, but by a largely extended shape of the nuclei (i.e. their elongated extensions).     

Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

Cell–cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.     

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges connecting spermatogenic cells in the black snail, Semisulcospira libertina (Mollusca, Mesogastropoda)

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges between spermatogenic cells are described in the black snail, Semisulcospira libertina. The bundles were occasionally observed in bridges connecting spermatogonia, spermatocytes and typical spermatids. Relations between bundles and centrioles could not be detected. The bundle had electron dense cross bands with a periodicity of approximately 200 nm, and attached to the membrane with almost right angle at the cross linker level. Phalloidin cytochemistry revealed that the bundle contained F-actin. In a case, a bundle connected two cytoplasmic bridges.     

Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts

We have used murine splenic erythrolasts infected with the anemia- inducing strain of Friend virus (FVA cells), as an in vitro model to study cytoskeletal elements during erythroid maturation and enucleation. FVA cells are capable of enucleating in suspension culture in vitro, indicating that associations with an extracellular matrix or accessory cells are not required for enucleation to occur. The morphology of FVA cells undergoing enucleation is nearly identical to erythroblasts enucleating in vivo. The nucleus is segregated to one side of the cell and then appears to be pinched off resulting in an extruded nucleus and reticulocyte. The extruded nucleus is surrounded by an intact plasma membrane and has little cytoplasm associated with it. Newly formed reticulocytes have an irregular shape, are vacuolated and contain all cytoplasmic organelles. The spatial distribution of several cytoskeletal proteins was examined during the maturation process. Spectrin was found associated with the plasma membrane of FVA cells at all stages of maturation but was segregated entirely to the incipient reticulocyte during enucleation. Microtubules formed cages around nuclei in immature FVA cells and were found primarily in the incipient reticulocyte in cells undergoing enucleation. Reticulocytes occasionally contained microtubules, but a generalized diffuse distribution of tubulin was more common. Vimentin could not be detected at any time in FVA cell maturation. Filamentous actin (F-actin) had a patchy distribution at the cell surface in the most immature erythroblasts, but F-actin bundles could be detected as the cells matured. F-actin was found concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts. Newly formed reticulocytes exhibited punctate actin fluorescence whereas extruded nuclei lacked F-actin. Addition of colchicine, vinblastine, or taxol to cultures of FVA cells did not affect enucleation. In contrast, cytochalasin D caused a complete inhibition of enucleation that could be reversed by washing out the cytochalasin D. These results demonstrate that F-actin plays a role in enucleation while the complete absence of microtubules or excessive numbers of polymerized microtubules do not affect enucleation.     

Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.     

Structure of actin paracrystals induced by nerve growth factor

When nerve growth factor is added to F-actin, well-ordered bundles of filaments are formed. These bundles are observed even at low concentrations of NGF²¹, but when N-bromosuccinimide-treated NGF, a biologically inactive form of the protein is used, a much higher concentration is required to produce aggregation. Moreover, the bundles induced by the modified NGF are not very well ordered and show amorphous aggregates attached at various points.Electron microscopy of paracrystals induced by native NGF shows that, although they resemble pure actin paracrystals induced by Mg²⁺, the interfilament spacing is larger and bridges connect the filaments. Optical diffraction patterns show, in addition to the off-meridional reflections characteristic of the actin helix, meridional reflections on the first and fourth layer-lines, at axial spacings of 37 and 9 nm. Measurements of the axial positions of the layer-lines show that the actin helical symmetry is not significantly different from that in pure actin paracrystals. The presence of the meridional reflections indicates that groups of two or three bridges with spacing 9 nm or nearly 9 nm are arranged along the bundles at a repeating interval of 37 nm.Actin filament bundles have been observed in several non-muscle cells, and specific actin-binding proteins have been identified as responsible for this aggregation. Our in vitro observations show that the biologically active form of NGF interacts with actin and organizes it into well-ordered paracrystalline arrays. The in vitro formation of NGF-actin complexes may be related to the in vivo mechanism of action of this growth factor.     

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

Summary The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.Abbreviations FITC fluorescein isothiocyanate - MF microfilament - MT microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline     

Modelling the dynamics of F-actin in the cell

The regulation of the interactions between the actin binding proteins and the actin filaments are known to affect the cytoskeletal structure of F-actin. We develop a model depicting the formation of actin cytoskeleton, bundles and orthogonal networks, via activation or inactivation of different types of actin binding proteins. It is found that as the actin filament density increases in the cell, a spontaneous tendency to organize into bundles or networks occurs depending on the active actin binding protein concentration. Also, a minute change in the relative binding affinity of the actin binding proteins in the cell may lead to a major change in the actin cytoskeleton. Both the linear stability analysis and the numerical results indicate that the structures formed are highly sensitive to changes in the parameters, in particular to changes in the parameter ϕ, denoting the relative binding affinity and concentration of the actin binding proteins.     

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34°C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34°C) or during a long-term heat treatment (30°C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34°C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34°C shock or during a continuous heat treatment at 30°C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34°C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30°C) using immunological detection of HSP70 in the cytoskeletal actin fraction.     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

Cytoskeletal interactions of synapsin I in non-neuronal cells

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.     

Mechanics of the F-actin cytoskeleton

Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is critical to numerous physical cellular processes, including cell adhesion, migration and division. Each of these processes require precise regulation of cell shape and mechanical force generation which, to a large degree, is regulated by the dynamic mechanical behaviors of a diverse assortment of F-actin networks and bundles. In this review, we review the current understanding of the mechanics of F-actin networks and identify areas of further research needed to establish physical models. We first review our understanding of the mechanical behaviors of F-actin networks reconstituted in vitro, with a focus on the nonlinear mechanical response and behavior of “active” F-actin networks. We then explore the types of mechanical response measured of cytoskeletal F-actin networks and bundles formed in living cells and identify how these measurements correspond to those performed on reconstituted F-actin networks formed in vitro. Together, these approaches identify the challenges and opportunities in the study of living cytoskeletal matter.     

Actomyosin is Involved in the Organization of the Microtubule Preprophase Band in Arabidopsis Suspension Cultured Cells

The microtubule preprophase bands （PPBs） participate in the sequence of events to position cell plates in most plants. However, the mechanism of PPB formation remains to be clarified. In the present study, the organization of PPBs in Arabidopsis suspension cultured cells was investigated by confocal laser scanning microscopy combined with pharmacological treatments of reagents specific for the cytoskeleton elements. Double staining of F-actin and microtubules （MTs） showed that actin filaments were arranged randomly and no colocalization with cortical MTs was observed in the interphase cells. However, cortical actin filaments showed colocalization with MTs during the formation of PPBs. A broad actin band formed with the broad MT band in the initiation of PPB and narrowed down together with the MT band to form the PPB. Nevertheless, broad MT bands were formed but failed to narrow down in cells treated with the F-actin disruptor latrunculin A. In contrast, in the presence of the F-actin stabilizer phalloidin, PPB formation did not exhibit any abnormality. Therefore, the integrity, but not the dynamics, of the actin cytoskeleton is necessary for the formation of normal PPBs. Treatment with 2, 3-butanedine monoxime, a myosin inhibitor, also resulted in the formation of broad MT bands, indicating that actomyosin may be involved in the rearrangement of MTs to form the PPBs. Double staining of MTs and myosin revealed that myosin concentrated on the PPB region during PPB formation. It is suggested that the actin cytoskeleton at the PPB site may serve as a rack to transport cortical MTs by using myosin when the broad MT band narrows down to form the PPB.