Protease-sensitive anchoring of microfilament bundles provides tracks for cytoplasmic streaming inVallisneria

Summary In mesophyll cells of the aquatic angiospermVallisneria gigantea Graebner, the endoplasm streams rotationally along the cell walls normal to the leaf surface in situ. Bundles of microfilaments anchored in the ectoplasm serve as tracks for the cytoplasmic streaming. In single mesophyll cells isolated by enzymatic digestion, hypertonic treatment induces abnormal streaming concomitant with plasmolysis, specifically at one or both of the shorter sides of an approximate rectangle. The disorderly arrangement of microfilaments in such cells has been confirmed by fluorescence microscopy of cells stained with FITC-phalloidin. While inhibitors of proteases added to the enzyme solution used for isolation of cells suppress the disturbance of rotational streaming, exogenously applied protease promotes it. The results suggest that bundles of microfilaments in the ectoplasm are stabilized by protease-sensitive factor(s) in the presence of the cell wall.     

Immobilisation of organelles and actin bundles in the cortical cytoplasm of the alga Chara corallina Klein ex. Wild

The mechanism by which sub-cortical actin bundles and membranous organelles are immobilised in the cortical cytoplasm of the alga Chara was studied by perfusing cells with a solution containing 1% Triton X-100. Light and scanning electron microscopy and the release of starch grains and chlorophyll-protein complexes indicated that the detergent extensively solubilised the chloroplasts. However, the sub-cortical actin bundles remained in situ even though they were originally separated from the plasma membrane by the chloroplasts. A fibrous layer between chloroplasts and plasma membrane became readily visible after detergent extraction of the cells and could be released by low-ionic-strength ethylenediaminetetraacetic acid, thioglycollate and trypsin. The same treatments applied to cells not subject to detergent extraction released the membrane-bound organelles and actin bundles and no fibrous meshwork was visible on subsequent extraction with Triton. It is, therefore, concluded that a detergent-insoluble cortical cytoskeleton exists and contributes to the immobility of the actin and cortical organelles in the cells.Abbreviation EDTA ethylenediaminetetraacetic acid     

Microtubule disassembly enhances reversible cytochalasin-dependent disruption of actin bundles in characean internodes

Summary Parallel bundles of actin filaments at the cortex-endoplasm interface provide tracks for myosin-generated cytoplasmic streaming in characean internodes. These bundles resist disassembly or structural modification when exposed to 10 μM cytochalasin D (CD) even though this concentration of CD rapidly (within minutes) but reversibly arrests streaming. Unexpectedly, we discovered that prolonged treatment with lower concentrations of CD could partially disassemble the subcortical actin bundles. Actin bundles became discontinuous following one- to several-day treatment with concentrations (6 μM) that reduced but did not arrest streaming, and the residual fragments mostly remained parallel to the chloroplast files. When microtubules were concurrently disassembled with tubulin-specific drugs, however, low CD concentrations (2.5–3 μM) completely arrested bulk streaming, disrupted the largely 2-dimensional actin bundle array and caused the formation of a coarse, thick-meshed actin network that extended from the cortex to the endoplasm. Despite such massive reconstruction, drug removal enabled cells to recover continuous parallel bundles and streaming. Recovery was possible if both or just one of the drugs were removed. In recovered cells, the streaming pattern frequently redeveloped in new directions that did not follow the chloroplast files, and later, chloroplast files readjusted to the new polarity established by the actin bundles. This first report on the complete and reversible disassembly of characean actin bundles provides new insights into the mechanism of actin bundle assembly and organization and supports the idea of indirect interactions between actin filaments and microtubules.     

Changes in Xenopus oocyte nucleus and cytoplasm organization after actin filaments depolymerization by latrunculin

Actin-containing filaments have been visualized inside the Xenopus oocyte nuclei due to combination of fluorescence and transmission electron microscopy. It has been shown that these filaments contact with nucleoli, spherical bodies and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing latrunculin causes membrane vesiculation in the cytoplasm, and disruption of the nucleoplasm and nuclear envelope integrity. We suppose that actin-containing filaments belong to crucial cell components which are involved in coordination of nuclear-cytoplasmic interactions as well as distribution and transport of intranuclear components in growing Xenopus oocytes.     

Membrane-associated actin filaments in the cortical cytoplasm of the rat mast cell

Cytoplasmic microfilaments are regular constituents of the cortical cytoplasm of rat mast cells. Heavy meromyosin binding to the microfilaments in glycerinated mast cells indicates that they represent actin filaments. Many of the actin filaments were found to be attached to spots of increased density of the plasma membrane. The actin filaments, possibly as part of an actomyosin system, may be involved in exocytosis of mast cell granules.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

Microtubule motor proteins and the organization of the pollen tube cytoplasm

Molecular motors are molecules that drive a wide range of activities (for example, organelle movement, chromosome segregation, and flagellar movement) in cells. Thus, they play essential roles in diverse cellular functions. Understanding their structures, mechanisms of action and different roles is therefore of great practical importance. The role of microtubules during pollen tube growth is presently not identified, nor are basic properties. We do not know, for example, where microtubules are organized, the extent of microtubule dynamics, and the polarity of microtubules in the pollen tube. Roles of microtubules and related motors in organelle trafficking are not clear. Regardless of scarce information, microtubule-based motors of both the kinesin and dynein families have been identified in the pollen tube. Most of these microtubule motors have also been found in association with membrane-bounded organelles, which suggest that these proteins could translocate organelles or vesicles along microtubules. The biochemical features of these proteins are typical of the motor protein class. Immunofluorescence microscopy of pollen tubes probed with antibodies that cross-react with microtubule motors indicate that these proteins are localized in different regions of the pollen tube; therefore, they could have different roles. Although a number of microtubule motors have been identified in the pollen tube, the role of these proteins during pollen tube germination and growth or organelle movement is not yet recognized, as tube elongation and organelle movement in the pollen tube depend mostly on actin filaments. In the effort to understand the specific role that microtubules and related motors have in the pollen tube, it is therefore necessary to identify the molecular machinery that interacts with microtubules. Furthermore, it is crucial to clearly establish the types of interaction between organelles and microtubules. This review summarizes the current state of the art on microtubule motors in the pollen tube, mainly surrounding the putative roles of microtubule motors in organelle movement and cytoplasmic organization. Some hypotheses and speculations are also presented.     

Microtubules at wound sites of Nitella internodal cells passively co-align with actin bundles when exposed to hydrodynamic forces generated by cytoplasmic streaming

The organization of cortical microtubules at wound sites in Nitella pseudoflabellata(A. Br. & Nordst.) em. R.D.W. and N. flexilis(L.) Ag. internodal cells was examined in relation to the regeneration of actin filament bundles in order to identify the mechanisms by which microtubules are oriented. Actin bundle regrowth occurs prior to that of microtubules, so it was considered possible that microtubule alignment is actin-dependent, perhaps mediated by cross-linking proteins. In all types of wounds investigated, subcortical actin bundles regenerated parallel to the direction of cytoplasmic streaming. Microtubule orientation patterns, however, varied according to the nature of wound formation and the type of wound wall eventually produced. In chloroplast-free windows induced by blue light irradiation, microtubule orientation varied according to the size of the window. Microtubules were randomized in 10- to 30-μm-wide windows where exposure to cytoplasmic flow is minimal, but were aligned more or less parallel to regenerated actin bundles in 80- to 100-μm-wide windows. Where co-alignment between microtubules and actin bundles was obvious after fluorescence labelling, electron micrographs revealed that microtubules and actin bundles were too widely spaced to account for any cross-linkages. Furthermore, treatments that inhibited or reduced cytoplasmic streaming without altering the direction of actin bundles caused randomization of microtubules previously oriented in the streaming direction, even in the presence of taxol. When evenly flat wound walls were induced by 10⁻⁴ M chlortetracycline, microtubules were co-aligned with nearby actin bundles at the surface of the wound wall. At wounds induced by treatment with 5 × 10⁻² M CaCl₂, however, microtubules were randomly oriented and preferentially located in the narrow clefts between the wound-wall protuberances, up to several micrometers away from the actin bundles near the wound-wall tips. These results indicate that microtubules regenerated in wounds are merely co-aligned with actin filament bundles because they are passively aligned by the hydrodynamic forces created by cytoplasmic flow. Received: 4 August 1998 / Accepted: 30 January 1999     

The structure of cortical cytoplasm

Actin-rich cortical cytoplasm of phagocytic leucocytes forms pseudopodia and controls cell shape and movement by generating directional propulsive and contractile forces. Proteins purified from leucocytes form and deform an actin matrix. Actin-binding protein (ABP) cross-links actin filaments into a three-dimensional lattice with perpendicular branches. This structure, which can be visualized in the electron microscope, is consistent with physical properties of actin-ABP matrices. Gelsolin binds one end of actin filaments with high affinity in the presence of calcium; acumentin, another protein, constitutively binds the other end with low affinity. Together these proteins can control actin filament length and thereby regulate expansion (propulsion) or collapse of the actin network. The assembly state of the network also controls myosin-based contractile forces. A tug-of-war decides the direction of lattice movement, regions of lesser structure tending to move toward regions of greater structure.     

Dynamics of actin filaments during tension-dependent formation of actin bundles

The actin cytoskeleton stress fiber is an actomyosin-based contractile structure seen as a bundle of actin filaments. Although tension development in a cell is believed to regulate stress fiber formation, little is known for the underlying biophysical mechanisms. To address this question, we examined the effects of tension on the behaviors of individual actin filaments during stress fiber (actin bundle) formation using cytosol-free semi-intact fibroblast cells that were pre-treated with the Rho kinase inhibitor Y-27632 to disassemble stress fibers into a meshwork of actin filaments. These filaments were sparsely labeled with quantum dots for live tracking of their motions. When ATP and Ca(2+) were applied to the semi-intact cells to generate actomyosin-based forces, actin meshwork in the protruded lamellae was dragged toward the cell body, while the periphery of the meshwork remained in the original region, indicating that centripetally directed tension developed in the meshwork. Then the individual actin filaments in the meshwork moved towards the cell body accompanied with sudden changes in the direction of their movements, finally forming actin bundles along the direction of tension. Dragging the meshwork by externally applied mechanical forces also exerted essentially the same effects. These results suggest the existence of tension-dependent remodeling of cross-links within the meshwork during the rearrangement of actin filaments, thus demonstrating that tension is a key player to regulate the dynamics of individual actin filaments that leads to actin bundle formation.     

Controlling the cortical actin motor

Actin is the essential force-generating component of the microfilament system, which powers numerous motile processes in eukaryotic cells and undergoes dynamic remodeling in response to different internal and external signaling. The ability of actin to polymerize into asymmetric filaments is the inherent property behind the site-directed force-generating capacity that operates during various intracellular movements and in surface protrusions. Not surprisingly, a broad variety of signaling pathways and components are involved in controlling and coordinating the activities of the actin microfilament system in a myriad of different interactions. The characterization of these processes has stimulated cell biologists for decades and has, as a consequence, resulted in a huge body of data. The purpose here is to present a cellular perspective on recent advances in our understanding of the microfilament system with respect to actin polymerization, filament structure and specific folding requirements.     

Mobility of filamentous actin in living cytoplasm

Filamentous actin in living cultured cells was labeled by microinjecting trace amounts of rhodamine-phalloidin (rh-pha) as a specific, high-affinity probe. The microinjection caused no detectable effect on cell morphology or cell division. The distribution of rh-pha- labeled filaments was then examined in dividing cells using image- intensified fluorescence microscopy, and the exchangeability of labeled filaments along stress fibers was studied during interphase using fluorescence recovery after photobleaching. rh-pha showed a rapid concentration at the contractile ring during cell division. In addition, recovery of fluorescence after photobleaching occurred along stress fibers with a halftime as short as 8 min. These observations suggest that at least some actin filaments undergo continuous movement and reorganization in living cells. This dynamic process may play an important role in various cellular functions.     

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.     

Disorganization of actin bundles of ectoplasmic specialization

The degradation of ectoplasmic specialization consisting of bundles of actin sandwiched between the plasma membrane and endoplasmic reticulum of the Sertoli cell, occurs just before spermiation. For elucidation of the processes involved in this degradation, changes in fibrous actin of the rat testis were analyzed using BODIPY-phalloidin by fluorescence and electron microscopy.
Before step 17, the fluorescence of BODIPY-phalloidin was evenly distributed around the spermatid head. When the spermatids became positioned at the luminal surface, the fluorescence had condensed on the concave side of the spermatid head. At step 19, lines of fluorescence distributed at regular intervals projected at right angles from the head. Ultrastructural observation showed that the tubulobulbar complex was formed at step 19 and electron-dense material accumulated around thin tubules of the tubulobulbar complex. Immunohistochemical examination of BODIPY-phalloidin showed that the electron dense materials around the thin tubules of the tubulobulbar complex had the capacity to bind to phallotoxin. Therefore the pattern of fluorescence in the spermatid at step 19 corresponds to that of the tubulobulbar complex.
Actin bundles of the ectoplasmic specialization would thus appear to de-polymerize into actin monomers via electron dense materials around the thin tubules of the tubulobulbar complex. The tubulobulbar complex may contribute to the disorganization of actin bundles.     

Short-term retention of actin filament binding proteins on lamellipodial actin bundles

Actin filaments are organised into sub-compartments of meshwork and bundles in lamellipodia. Localisation of fascin, the LIM and SH3 domain protein 1 (lasp-1), and lasp-2 to the bundles suggest their involvement in that organisation; however, their contributions remain unclear. We have compared the turnover of these proteins with actin at the bundle. After photobleaching, EGFP-actin recovered inwards from the bundle tip, consistent with the retrograde flow by treadmilling. In contrast, the recovery of EGFP-fascin, -lasp-1 and -lasp-2 occurred from the anterograde direction. These results suggest that these molecules would participate in the stabilisation of bundles but not in initiation.     

Thickness distribution of actin bundles in vitro

Bundles of filamentous actin form the primary building blocks of a broad range of cytoskeletal structures, including filopodia, stereocilia and microvilli. In each case, the cell uses specific associated proteins to tailor the dynamics, dimensions and mechanical properties of the bundles to suit a specific cellular function. While the length distribution of actin bundles was extensively studied, almost nothing is known about the thickness distribution. Here, we use high-resolution cryo-TEM to measure the thickness distribution of actin/fascin bundles, in vitro. We find that the thickness distribution has a prominent peak, with an exponential tail, supporting a scenario of an initial fast formation of a disc-like nucleus of short actin filaments, which only later elongates. The bundle thicknesses at steady state are found to follow the distribution of the initial nuclei indicating that no lateral coalescence occurs. Our results show that the distribution of bundles thicknesses can be controlled by monitoring the initial nucleation process. In vivo, this is done by using specific regulatory proteins complexes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stimulation of endocytosis and actin dynamics by Oskar polarizes the Drosophila oocyte

In Drosophila, localized activity of oskar at the posterior pole of the oocyte induces germline and abdomen formation in the embryo. Oskar has two isoforms, a short isoform encoding the patterning determinant and a long isoform of unknown function. Here, we show by immuno-electron microscopy that the two Oskar isoforms have different subcellular localizations in the oocyte: Short Oskar mainly localizes to polar granules, and Long Oskar is specifically associated with endocytic membranes along the posterior cortex. Our cell biological and genetic analyses reveal that Oskar stimulates endocytosis, and that its two isoforms are required to regulate this process. Furthermore, we describe long F-actin projections at the oocyte posterior pole that are induced by and intermingled with Oskar protein. We propose that Oskar maintains its localization at the posterior pole through dual functions in regulating endocytosis and F-actin dynamics.