State of actin during the cycle of cohesiveness of the cytoplasm in parthenogenetically activated sea urchin egg

By the DNase I inhibition assay it is shown that the cytoplasmic matrix isolated 60 min after procaine activation of Paracentrotus lividus eggs contains about 20% of the total egg actin, mostly in polymerized form (85%). Electron microscopy studies on this cytoplasmic structure after treatment with heavy meromyosin (HMM), reveal that the decorated actin filaments are organized in bundles which are distributed radially, with the arrowheads pointing towards the central region. In addition few microtubules and a network of non-decorated microfilaments of about 3 nm diameter are observed. From the cytoplasmic pH determination and the DNase I inhibition assay on homogenates of eggs which were taken at different times of activation, it cannot be inferred that a direct relationship between the increase in the cytoplasmic pH and the increase in the amount of polymerized actin or of cytoplasmic matrix exists. Activation experiments carried out in the presence of colchicine shows that, although the formation of the cytoplasmic matrix is inhibited, polymerization of actin still occurs. Moreover, from the inhibition effects of cytochalasin B (CB) added before the activator it is shown that polymerization of actin is a necessary step for the organization of the cytoplasmic matrix. However, the cycles of cohesiveness of the cytoplasm observed in the course of the activation process do not appear to depend on cycles of polymerization and depolymerization of actin.     

Influence of the polyamine spermine on the organization of cortical filaments in isolated cortices of Xenopus laevis eggs

Microinjection of spermine into Xenopus laevis eggs induces precocious furrowing, and together with known spermine-actin interactions, suggests that spermine may be affecting cortical microfilaments involved in cytokinesis. An electron microscopic study of injected eggs revealed that the ultrastructure of the induced furrows was similar to that of both artificially activated eggs and fertilized eggs. In isolated egg cortices, increasing spermine concentrations (1, 3 and 10 mM) resulted in marked changes in cortical microfilament organization. At low concentrations, spermine appeared to stabilize microfilaments and at higher concentrations induced lateral associations between filaments and formation of bundles. The actin nature of these cortical microfilaments was confirmed by immunocytochemistry. The electrophoretic profiles of proteins from control and spermine-treated isolated cortices were similar. Although the total protein content of isolates in 3 and 10 mM spermine was elevated, the relative actin content remained constant. The results are in agreement with previous in vitro studies of polyamine interactions with actin and support the hypothesis that a polyamine-actin interaction may be important in the regulation of cytokinesis.     

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

Dual effect of procaine in sea urchin eggs. Inducer and inhibitor of microtubule assembly

An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin.     

The cortical cytoskeleton and its role in sperm penetration of the mammalian egg

In this study isolated cortical regions of both penetrated and nonpenetrated Syrian hamster eggs were examined in whole mounts and platinum replicas of detergent-extracted cortical patches. Two types of cytoskeletal organization were observed in the egg cortex: Loose networks (LN regions) with integrated localized dense networks (LDN regions). Decoration with heavy meromyosin and labeling with antiactin/protein G gold both indicate that the cortical cytoskeleton consists mainly of a LN of actin microfilaments and several types of nonactin filaments, whereas LDN regions dispersed within the LN were comprised of nonactin filaments. Cortical patches and replicas of eggs incubated with sperm for 10-15 min provide evidence that cortical microfilaments may be intimately associated with penetrating spermatozoa. The results of this investigation provide the first high resolution view of the cortical cytoskeletal domain of a mammalian egg and suggest that actin microfilaments might play a role in sperm penetration of the egg cortex.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Effects of phytohormones on the cytoskeleton of the plant cell

This review highlights the effects of ??classic?? phytohormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene, and brassinosteroids) and also of important signaling molecules, such as jasmonic acid, strigolactones, and nitric oxide, on the main components of the plant cytoskeleton, microtubules and microfilaments. The effects of these growth regulators on orientation and organization of microtubules and actin filaments, realization of cytoskeleton-dependent processes, expression of tubulin and actin genes, and interaction of various phytohormones in their influence on the cytoskeleton are discussed.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

Secondary lysosomes as an integral part of the cytoskeleton: a morphological study in rat Kupffer cells

Rat Kupffer cells contain the three major cytoskeletal components: microfilaments (MF), microtubules (MT), and intermediate filaments (IF) of the vimentin type. Previous cytomagnetometric data obtained from alveolar macrophages and rat Kupffer cells in culture provided evidence that actin filaments contribute to the movements of lysosomes. The lysosomal transport in living cells was affected, when the MFs were selectively disturbed, whereas the depolymerization of the MTs had no effect on the lysosomal movement measured by cytomagnetometric means. Immunofluorescence and ultrastructural studies of isolated and cultured rat Kupffer cells, presented in this paper, will investigate the relationship between lysosomes and the cytoskeleton. The principal filamentous structure in the peripheral cytoplasm of Kupffer cells in a dense meshwork of actin filaments. The dimension of the meshes combined with the dimensions of lysosomes implies the necessity of either (i) disintegration of the actin filament cross-links, (ii) depolarymerization and redistribution of MF's, or (iii) a displacement of actin filaments by the lysosomes during the organelle transport. The presence of microtubules in cytoplasmic protrusions and their track from the periphery to the perinuclear region during interphase might play a role in the transport mechanism of lysosomes, the more so because microtubules could often be demonstrated in closest association with lysosomes even in the first phase of endocytosis. The distribution pattern of vimentin, found as a dense interconnected framework surrounding the lysosomes like a basket, could play a role in positioning the organelles. The dynamic functions of MF's and MT's and their multifunctionality led to an adaptive and flexible organization of these filaments which may both be involved in lysosomal motion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

Redundant mechanisms recruit actin into the contractile ring in silkworm spermatocytes

Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation). Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation). To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex). In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.     

A plant-specific kinesin binds to actin microfilaments and interacts with cortical microtubules in cotton fibers

A novel kinesin, GhKCH1, has been identified from cotton (Gossypium hirsutum) fibers. GhKCH1 has a centrally located kinesin catalytic core, a signature neck peptide of minus end-directed kinesins, and a unique calponin homology (CH) domain at its N terminus. GhKCH1 and other CH domain-containing kinesins (KCHs) belong to a distinct branch of the minus end-directed kinesin subfamily. To date the KCH kinesins have been found only in higher plants. Because the CH domain is often found in actin-binding proteins, we proposed that GhKCH1 might play a role in mediating dynamic interaction between microtubules and actin microfilaments in cotton fibers. In an in vitro actin-binding assay, GhKCH1's N-terminal region including the CH domain interacted directly with actin microfilaments. In cotton fibers, GhKCH1 decorated cortical microtubules in a punctate manner. Occasionally GhKCH1 was found to be associated with transverse-cortical actin microfilaments, but never with axial actin cables in cotton fibers. Localization of GhKCH1 on cortical microtubules was independent of the integrity of actin microfilaments. Thus, GhKCH1 may play a role in organizing the actin network in coordination with the cortical microtubule array. These data also suggest that flowering plants may employ unique KCHs to coordinate actin microfilaments and microtubules during cell growth.