Microtubule and microfilament organization in maturing human oocytes

Various stages of immature human oocytes were imaged for microtubule, microfilament and chromatin organization. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. The asters appeared to elongate and encompass the condensed chromatin. At metaphase I stage, microtubules were detected in the meiotic spindle. The meiotic spindle in metaphase II was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. After germinal vesicle breakdown, treatment with taxol induced numerous cytoplasmic foci of microtubules, mainly in the cortex of the oocyte. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found near the germinal vesicle position. After germinal vesicle breakdown, the microfilaments were seen in both the cortex and around the female chromatin. In conclusion, this study suggests that both microtubules and microfilaments are closely associated with the reconstruction and proper positioning of chromatin after germinal vesicle breakdown and during meiotic maturation in human oocytes.     

Structural changes in oocyte nucleoli of Xenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II-III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II-III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II-III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4',6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II-III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble.     

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms

Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.     

Unipolar microtubule array is directly involved in nurse cell-oocyte transport

The telotrophic ovariole of Rhodnius prolixus is richly endowed with microtubules (MTs). An extensive, stable array of MTs packs the trophic core and trophic cords which link the nurse cell compartments to the growing oocytes. This system is excellent to study MT-based transport as the MTs are believed to play a role in transport of nurse cell-produced mitochondria, ribosomes, and mRNAs to the oocytes. We investigated MT polarity and molecular MT motors in this unidirectional transport system. Hook decoration revealed that the MTs of the trophic core and cords have their plus (+) ends in the tropharium and minus (-) ends in the oocytes. Video differential interference optics (DIC) microscopy showed that vesicle transport was saltatory, ATP-dependent, and had an average velocity of 0.77 micron/sec toward the oocyte. Transport was sensitive to 2 mM N-ethylmaleimide (NEM) and 50 microM vanadate and resistant to 1 mM 5'-adenylylimidodiphosphate (AMP-PNP) and 5 microM vanadate. We report that the unipolar, acetylated trophic cord MTs play a direct role in nurse cell-oocyte transport via a cytoplasmic dynein-like retrograde motor.     

Patterns of localization and cytoskeletal association of two vegetally localized RNAs, Vg1 and Xcat-2

In Xenopus, localization of a rare class of mRNAs during oogenesis is believed to initiate pattern formation in the early embryo. We have determined the pattern of RNA localization for one of these RNAs, Xcat-2, which encodes a putative RNA-binding protein related to Drosophila nanos (Mosquera, L., Forristall, C., Zhou, Y. and King, M. L. (1993) Development 117, 377-386). Xcat-2 is exclusively localized to the mitochondrial cloud in stage I oocytes, moves with this body into the vegetal cortex during stage II and, later, partitions into islands consistent with it being a component of the germ plasm. As previously shown, Vg1 is not localized to the vegetal cortex until stage IV and distributes to all vegetal blastomeres during development. We found a direct correlation between the localized condition of these RNAs and their recovery in a detergent-insoluble fraction. We present evidence suggesting that differential RNA binding to a cytoskeletal component(s) in the vegetal cortex determines the pattern of inheritance for that RNA in the embryo.     

In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway

Anterior patterning of the Drosophila embryo depends on localization of bicoid (bcd) mRNA to the anterior pole of the developing oocyte, and bcd mRNA localization requires both the exuperantia (exu) gene and an intact microtubule cytoskeleton. To gain insight into the mechanism of anterior patterning, we have used time lapse laser scanning confocal microscopy to analyze transport of particles containing a Green Fluorescent Protein-Exu fusion (GFP-Exu), and to directly image microtubule organization in vivo. Our observations indicate that microtubules are required for three forms of particle movement within the nurse cells, while transport through the ring canals linking the nurse cells and oocyte appears to be independent of both microtubules and actin filaments. As particles enter the oocyte, a final microtubule-dependent step directs movement to the oocyte cortex. However, our observations and previous studies suggest that the polarity of the oocyte microtubule network is not in itself sufficient to generate anterior asymmetry, and that additional factors are required to restrict morphogens to the anterior pole. Based on these observations, we propose a multi-step anterior localization pathway.     

Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes

To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10,000 g for 5 min at 20 degrees C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100-300 microns, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between -35 and -65 mV, and the membrane resistances were in the 500 k omega to 1 M omega range. Under voltage clamp conditions, the oocytes exhibited Ca(2+)-activated Cl- currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.     

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.     

Fatal meconium aspiration in spite of appropriate perinatal airway management: pulmonary and placental evidence of prenatal disease

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.     

Localized maternal proteins in Xenopus revealed by subtractive immunization

It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.     

Localization of activin and follistatin proteins in the Xenopus oocyte

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos.     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.     

Modified mRNA rescue of maternal CK1/8 mRNA depletion in Xenopus oocytes

This work addresses two issues, the use of antisense oligodeoxynucleotides to deplete specific mRNAs in Xenopus oocytes to analyze their functions during development and the role of cytokeratin filaments in cells of the early Xenopus embryo. We have shown previously that depletion of cytokeratin CK1/8 mRNA causes defects in the early embryo. In this study, we show that the oligos, modified with phosphoramidate linkages to improve stability, are capable of degrading exogenous mRNA up to 27 hours after injection in the oocyte. For this reason, the phenotype could not be rescued by injection of a synthetic CK1/8 mRNA. However, modification of the synthetic CK1/8 mRNA, which prevents annealing of the antisense oligonucleotide used for depleting the endogenous CK1/8 mRNA, did result in the rescue of the CK1/8 depletion phenotype. These results demonstrate that the phenotype observed after depletion of the CK1/8 mRNA is truly caused by the lack of CK1/8 protein. Injection of the closely related type II cytokeratin (CK55) did not result in the same level of rescue of the CK1/8 depletion phenotype, suggesting that structurally similar members of the cytokeratin family, expressed at different stages of development, cannot substitute for each other in the early embryo.     

Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum

In many organisms, pattern formation in the embryo develops from the polarized distributions of messenger RNAs (mRNAs) in the egg. In Xenopus, the mRNA encoding Vg1, a growth factor involved in mesoderm induction, is localized to the vegetal cortex of oocytes. A protein named Vera was shown to be involved in Vg1 mRNA localization. Vera cofractionates with endoplasmic reticulum (ER) membranes, and endogenous Vg1 mRNA is associated with a subcompartment of the ER. Vera may promote mRNA localization in Xenopus oocytes by mediating an interaction between the Vg1 3' untranslated region and the ER subcompartment.     

Female genital mutilation: an overview

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Provisional bilateral symmetry in Xenopus eggs is established during maturation

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.     

Chemical modification and chemical cross-linking for protein/enzyme stabilization

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.     

Metabolism of oocyte construction and the generation of histospecificity in the cleaving egg. Lessons from nereid annelids

The growing oocyte and the developing egg of nereid polychaetes are easily accessible to observation and experimental work, a precondition for our research. In preparation for a single semelparous act of reproduction, nereid females reutilize somatic biomass for the synchronized production of numerous oocytes. To keep oogenesis going somatic resources become recycled by the eleocytes and are supplied to the oocytes in form of vitellogenin and nucleotides (among other identified and yet unidentified substances). Both oocytes and eleocytes are free-floating coelomic cells. We postulate that availability of metabolites produced by the eleocytes might suffice to drive synchronous oocyte growth. The cortex of the fully differentiated oocyte contains numerous cortical granules which after fertilization empty by exocytosis thus causing a profound structural reorganization of the zygote cortex. Early development of nereids is extremely constant in time and spatial pattern and from the onset cleavages create diversity among the blastomeres. We have documented a correlation between the quality and amount of cytoplasm, the cell cycle duration and the histogenetic fate of such blastomeres. Experimental change of cytoplasmic proportions of early cleavage cells has serious consequences for axial development. Using a number of differentiation markers we were able to analyze the necessity of certain cleavage steps for the acquisition of the determined state.     

Structural and endocrine aspects of equine oocyte maturation in vivo

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.     

Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules

In activated sea urchin coelomocytes, cytoplasmic organelles move along distinct actin and microtubule dependent pathways, actin-based motility is driven by an unconventional myosin, and microtubule disassembly does not effect actin-dependent organelle motility [D'Andrea et al., 1994: J. Cell Sci. 107:2081-2094]. Given the growing evidence for potential interactions between components of the actin and microtubule cytoskeletons, we examined the effect of actin filament disassembly on the movement of mitochondria along microtubules in activated coelomocytes. Coelomocytes treated with cytochalasin B (CB), to disrupt actin filaments, exhibited a thinning of the cytoplasm, enhanced lateral undulation of microtubules, and ceased centripetal cortical flow of actin. Interestingly, the loss of actin filaments resulted in a approximately 1.5-fold increase in the average velocity of outward and inward moving mitochondria and increased the frequency of centripetal movement. To test if enhanced motility along microtubules was a consequence of decreased actin-myosin interaction, coelomocytes were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin activity [Cramer and Mitchison, 1995: J. Cell Biol. 131:179-189]. BDM inhibited all types of actin-based motility observed in these cells including retrograde cortical flow, protrusion and retraction of the cell edge, and movement of intracellular organelles. Surprisingly, BDM treatment stopped the movement of mitochondria in CB-exposed cells, suggesting that BDM can also act as an inhibitor of organelle movement along microtubules. Collectively, these data demonstrated that microtubule-dependent mitochondrial motility and microtubule movement were sensitive to changes in the assembly state of the actin cytoskeleton.