scholarly journals Actin filament content and organization in unstimulated platelets.

1984 ◽  
Vol 98 (6) ◽  
pp. 1985-1991 ◽  
Author(s):  
J E Fox ◽  
J K Boyles ◽  
C C Reynolds ◽  
D R Phillips
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Gel Filtration ◽  
Structural Reorganization ◽  
Filamentous Form ◽  
Thin Sections ◽  
Cell Extracts ◽  
Triton X ◽  
Filament Content

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.

scholarly journals Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage.

1985 ◽  
Vol 101 (4) ◽  
pp. 1463-1472 ◽  
Author(s):  
J Boyles ◽  
J E Fox ◽  
D R Phillips ◽  
P E Stenberg
Keyword(s):  
Actin Filaments ◽  
Structural Organization ◽  
Human Platelet ◽  
Gel Filtration ◽  
Human Platelets ◽  
Thin Sections ◽  
Whole Cells ◽  
Dense Cytoplasm ◽  
Cross Links ◽  
Triton X

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

scholarly journals Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

2002 ◽  
Vol 156 (6) ◽  
pp. 1065-1076 ◽  
Author(s):  
Shoichiro Ono ◽  
Kanako Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Biological Properties ◽  
Actin Dynamics ◽  
Background Suppression ◽  
Thin Filaments ◽  
Actin Organization ◽  
C Elegans ◽  
Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

scholarly journals F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

1996 ◽  
Vol 135 (5) ◽  
pp. 1291-1308 ◽  
Author(s):  
L G Tilney ◽  
P Connelly ◽  
S Smith ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Modular Design ◽  
Thin Sections ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Phalloidin Staining ◽  
Filament Bundles ◽  
End To End ◽  
As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

scholarly journals A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

1984 ◽  
Vol 99 (3) ◽  
pp. 994-1001 ◽  
Author(s):  
H Hosoya ◽  
I Mabuchi
Keyword(s):  
Sea Urchin ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Initial Rate ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Capping Protein ◽  
Sea Urchin Egg ◽  
Rate Of Polymerization ◽  
Hemicentrotus Pulcherrimus

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

scholarly journals The actin filament content of hair cells of the bird cochlea is nearly constant even though the length, width, and number of stereocilia vary depending on the hair cell location.

1988 ◽  
Vol 107 (6) ◽  
pp. 2563-2574 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney
Keyword(s):  
Hair Cell ◽  
Actin Filament ◽  
Cross Sections ◽  
Hair Cells ◽  
Actin Filaments ◽  
Membrane Surface ◽  
Scanning Electron Micrographs ◽  
Length Width ◽  
Filament Content ◽  
Filament Bundle

By direct counts off scanning electron micrographs, we determined the number of stereocilia per hair cell of the chicken cochlea as a function of the position of the hair cell on the cochlea. Micrographs of thin cross sections of stereociliary bundles located at known positions on the cochlea were enlarged and the total number of actin filaments per stereocilium was counted and recorded. By comparing the counts of filament number with measurements of actin filament bundle width of the same stereocilium, we were able to relate actin filament bundle width to filament number with an error margin (r2) of 16%. Combining this data with data already published or in the process of publication from our laboratory on the length and width of stereocilia, we were able to calculate the total length of actin filaments present in stereociliary bundles of hair cells located at a variety of positions on the cochlea. We found that stereociliary bundles of hair cells contain 80,000-98,000 micron of actin filament, i.e., the concentration of actin is constant in all hair cells with a range of values that is less than our error in measurement and/or biological variation, the greatest variation being in relating the diameters of the stereocilia to filament number. We also calculated the membrane surface needed to cover the stereocilia of hair cells located throughout the cochlea. The values (172-192 micron 2) are also constant. The implications of our observation that the total amount of actin is constant even though the length, width, and number of stereocilia per hair cell vary are discussed.

scholarly journals Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

1981 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
S Sato ◽  
M Nakao
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Thin Sections ◽  
Fixation Treatment ◽  
Human Erythrocyte Membranes ◽  
Inside Out

Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid- glutaraldehyde fixation. Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted more than 90 percent of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles. When such spectrin-depleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles. The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form a irregular but continuous meshwork parallel to the membrane. Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles. However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin- or purified spectrin- reassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components. Two modes of association of actin filaments with the membrane were seen: end-to-membrane and side-to- membrane associations. In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane.

scholarly journals 110OSMOTIC STRESS ON THE CELLULAR ACTIN FILAMENT ORGANIZATION OF IN VITRO PRODUCED PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 177 ◽  
Author(s):  
H. Men ◽  
Y. Agca ◽  
S.F. Mullen ◽  
E.S. Critser ◽  
J.K. Critser
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Developmental Competence ◽  
Tween 20 ◽  
Embryonic Cells ◽  
Sucrose Solutions ◽  
The Status ◽  
Osmotic Treatment ◽  
Triton X

Disruption of the actin cytoskeleton is one of the leading causes in low survival of pig embryos after cryopreservation (Dobrinsky et al., 2000 Biol. Reprod. 62, 564–570). In this study, the effect of osmotic stress on cellular actin filament organization in porcine embryos produced in vitro was studied. Excellent quality Day 6 (fertilization=Day 0) porcine blastocysts were randomly exposed to 6 different anisosmotic sucrose solutions (75, 150, 210, 600, 1200, 2400mOsm) for 10min. Embryos were then returned to embryo culture medium (NCSU-23) after washing with NCSU-23, and cultured under 38.5°C, 5% CO2 in air with maximal humidity for them to recover. Blastocysts cultured in NCSU-23 medium (280mOsm) served as a control for embryos with intact actin filament organization. Blastocysts treated with 7.5μgmL−1 cytochalasin-b for 60min served as a control for embryos with F-actin depolymerization. Eighteen hours post-anisosmotic treatments, all blastoysts were fixed in 3.7% paraformaldehyde in PBS for 60min and stored in PBS with 0.1% Triton X-100 and 0.2% sodium azide at 4°C. Staining of actin filaments was performed according to procedures described earlier (Wang et al., 1999 Biol. Reprod. 60, 1020–1028). Embryos were blocked in PBS with 20mgmL−1 BSA and 150mM glycine for 30min. After being washed in PBS with 0.1% Tween 20 for 60min, embryos were stained with 10UmL−1 Alexa Fluor 488 phalloidin in PBS with 0.1% Tween 20 at 38.5°C for 60min, and then washed twice in PBS with 0.1% Tween 20 for 60min each. The status of actin filaments in embryonic cells was examined by confocal microscopy. Integrity of cellular actin filaments was classified as either intact or disrupted according to the distribution within embryonic cells. Blastocysts were then classified according to the status of actin filaments in embryonic cells. Data were analyzed using logistic regression. Results from 7 replicates are displayed in Table 1. There was a significant relationship between osmotic treatment levels and the probability of blastocysts with disrupted cellular actin filaments (P<0.0001). These data support the hypothesis that porcine embryos are very sensitive to osmotic changes. Ongoing experiments will assess the extent of actin disruption required to significantly reduce developmental competence of pig blastocysts. This study was supported by Monsanto Company. Table 1 Cellular actin filament integrity of in vitro produced porcine blastocysts after being treated with sucrose solutions with different osmolalities (mOsm)

scholarly journals Actin, microvilli, and the fertilization cone of sea urchin eggs.

1980 ◽  
Vol 87 (3) ◽  
pp. 771-782 ◽  
Author(s):  
L G Tilney ◽  
L A Jaffe
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Germinal Vesicle ◽  
Rapid Phase ◽  
Thin Sections ◽  
Step Process ◽  
Sea Urchin Eggs ◽  
Early Stages

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.

scholarly journals Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

2009 ◽  
Vol 20 (8) ◽  
pp. 2160-2173 ◽  
Author(s):  
Colleen T. Skau ◽  
Erin M. Neidt ◽  
David R. Kovar
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Animal Cells ◽  
Contractile Ring ◽  
Direct Role ◽  
Ring Assembly

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

scholarly journals Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

1996 ◽  
Vol 133 (1) ◽  
pp. 61-74 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Nurse Cells ◽  
Ring Canal ◽  
Contractile Ring ◽  
Thin Sections ◽  
Development Stages ◽  
Ring Canals ◽  
Filament Bundles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

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