Redistribution Of Microtubules Research Articles

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

Microtubule Redistribution in Growth Cones Elicited by Focal Inactivation of Kinesin-5

In order for growth cones to turn, microtubules from the central domain must preferentially invade the peripheral domain in the direction of the turn. Recent studies suggest that kinesin-5 (also called Eg5 or kif11) suppresses the invasion of microtubules into the peripheral domain on the side of the growth cone opposite the direction of turning. In theory, kinesin-5 could elicit these effects by acting on the microtubules within the peripheral domain itself, by acting on microtubules in the central domain, or in the transition zone between these two domains. In rat neurons expressing kinesin-5, we documented the presence of kinesin-5 in both domains of the growth cone and especially enriched in the transition zone. We then focally inactivated kinesin-5 in various regions of the growth cone, using micro-chromophore-assisted laser inactivation. We found that a greater invasion of microtubules into the peripheral domain occurred when kinesin-5 was inactivated specifically in the transition zone. However, there was no effect on microtubule invasion into the peripheral domain when kinesin-5 was inactivated in the peripheral domain itself or in the central domain. In other experiments, frog growth cones were observed to turn toward a gradient of a drug that inhibits kinesin-5, confirming that asymmetric inactivation of kinesin-5 can cause the growth cone to turn. Finally, expression of a phospho-mutant of kinesin-5 resulted in greater microtubule invasion throughout the peripheral domain and an inhibition of growth cone turning, implicating phosphorylation as a means by which kinesin-5 is regulated in the growth cone.

Expressing murine p56Hckca promotes HeLa cells’ motility and invasion via triggering redistribution of F-actin and microtubules

Hck is the unique example among the Src PTKs to be expressed as two isoforms, which are generated by alternative translation. The two isoforms differs from each other by a 21 N-terminal amino acids sequence which supports myristoylation. Though it has been shown that these different acylation states govern the different subcellular localization of the isoforms and each Hck isoform could play a specific role, little study focus on the function of p56Hck. To investigated the role of p56Hck isoform in cell migration, GFP targeted p56Hck plasmid and its constitutively active form were constructed and transiently transfected into HeLa cells, F-actin staining and Indirect immunofluorescence for microtubules were then performed. Phagokinetic track motility assay and In vitro invasion assays were also investigated after transiently transfection respectively. In this study, we found ectopically expressing a constitutively active form of 56Hck will lead to membrane protrusion and F-actin reorganization in HeLa cells. Both 56Hck and its constitutive active form will lead to redistribution of microtubules and enhancement of cell motility and cell invasion. Hck inhibitor PP2 supplementation eliminated cell motility and cell invasion of p56Hck while PP3, a negative control of PP2 didn't eliminate cell motility and cell invasion of p56Hck. It is indicated that enhanced cell motility and cell invasion in p56Hck ectopically expressed HeLa cells are the results of reorganization of F-actin and microtubules.

Paclitaxel Induces Apoptosis Through Activation of Nuclear Protein Kinase C-δ and Subsequent Activation of Golgi Associated Cdk1 in Human Hormone Refractory Prostate Cancer

Emerging evidence shows that the translocation of apoptosis related factors on cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus and nucleus, has a crucial role in the apoptotic process. We characterized the effect of paclitaxel (Sigma®) on Golgi involved apoptosis in human hormone refractory prostate cancer. FACScan™ flow cytometric analysis was used to determine cell cycle distribution and the subG1 (apoptosis) population. Protein expression and localization were detected by Western blot, confocal microscopic examination and the sucrose gradient separation technique. Paclitaxel induced Golgi apparatus disassembly and interaction between Golgi complexes and mitochondria. Discontinuous sucrose gradient fractionation was used to determine and collect Golgi containing fractions. Data revealed that paclitaxel induced an increase of Cdk1 activity and DR5 expression on the Golgi complex that was associated with increased cleavage of caspase-8, a DR5 downstream factor, and caspase-3 into catalytically active fragments. Data were validated by confocal immunofluorescence microscopy. Golgi associated effects were inhibited by the Cdk1 inhibitor roscovitine (Sigma), suggesting a critical role for Golgi-Cdk1. Also, paclitaxel caused an increase of nuclear but not of Golgi associated PKC-δ activity. The selective PKC-δ inhibitor rottlerin (Sigma) completely inhibited the increase of Golgi-Cdk1 activity, suggesting that nuclear PKC-δ served as an upstream regulator of Golgi-Cdk1. Data suggest that paclitaxel induces nuclear translocation and activation of PKC-δ, which in turn causes Golgi-Cdk1 activation, leading to Golgi associated DR5 up-regulation, and caspase-8 and 3 activation. Golgi mediated signaling cascades facilitate mitochondria involved apoptotic pathways and at least partly explain the anticancer activity of paclitaxel action.

Angiotensin II induces microtubule reorganization mediated by a deacetylase SIRT2 in endothelial cells

Angiotensin II has been implicated in vascular remodeling. Microtubule composed of tubulins regulates cell shape, migration and survival. Tubulin acetylation has an important role in the control of microtubule structure and microtubule-based cellular functions. In this study, angiotensin II induced disassembly and deacetylation of α-tubulin, which were blocked by pretreatment with an angiotensin II type 1 receptor blocker losartan and a sirtuin class deacetylase inhibitor sirtinol, and by depletion of a deacetylase SIRT2 using RNA interference. We investigated the involvement of SIRT2 in angiotensin II-induced endothelial cell migration using the Boyden chamber method. Angiotensin II caused a significant increase in cell migration, which was blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported that angiotensin II is involved in cytoskeletal reorganization stimulated by mechanical stretch in endothelial cells. We also demonstrated that endothelial cells subjected to a 10% uniaxial stretch showed vertical alignment to the direction of tension and tubulin deacetylation in the peripheral side of cells, in comparison with control static cells. The mechanical stretch-induced changes of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion. Immunofluorescence microscopy showed that acetylated tubulin was decreased in platelet-endothelial cell adhesion molecule-1-positive cells in the intima of the aortic walls in mice loaded with angiotensin II, in comparison with mice loaded with control vehicle. These data show that angiotensin II and mechanical stretch stimulate microtubule redistribution and deacetylation via SIRT2 in endothelial cells, suggesting the emerging role of SIRT2 in hypertension-induced vascular remodeling.

Abstract 4848: Angintensin II Stimulates Microtubule Reorganization Mediated by SIRT2 in Endothelial Cells

Angiotensin II has been implicated in the pathophysiology of vascular remodeling. Microtubule composed of tubulins regulates cell shape, migration and survival. Tubulin acetylation plays an important role in the control of microtubule structure and microtubule-based cellular functions. We studied angiotensin II-induced changes in microtubules in endothelial cells. Immunofluorescence microscopy showed that acetylated tubulin was decreased in PECAM-1-positive cells in the intima of the aortic walls in mice loaded with angiotensin II, compared to mice loaded with control vehicle. In cultured human endothelial cells in quiescence, α -tubulin-positive microtubules were acetylated and showed radial layout from microtubule-organizing center to peripheral edge of cells. Angiotensin II induced disassembly and deacetylation of α -tubulin, which were blocked by pretreatment with losartan that is an angiotensin II type 1 receptor blocker, sirtinol that is an inhibitor for sirtuin class deacetylases, and depletion of a deacetylase SIRT2 by RNA interference. We investigated the involvement of SIRT2 in angiotensin II-induced endothelial cell migration using Boyden chamber method. Angiotensin II caused a significant increase in cell migration, which was blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported that angiotensin II is involeved in cytoskeletal reorganization stimulated by mechanical stretch in endothelial cells. To investigate the effects of mechanical stretch on microtubule reorganization, cultured endothelial cells were subjected to 10% uniaxial stretch and examnied by immunofluorescence. Mechanically stretched cells showed vertical alignment to tension direction and tubulin deacetylation in peripheral side of cells, compared to control static cells. Mechanical stretch-induced changes of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion. These data suggest that angiotensin II and mechanical stretch stimulate microtubule redistribution and deacetylation via SIRT2 in endothelial cells. Further studies are necessary to elucidate the roles of SIRT2-mediated microtubule reorganization in vascular remodeling.

Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host–pathogen interactions.

Coordination of Actin Filament and Microtubule Dynamics during Neurite Outgrowth

Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.

Live‐cell analysis of mitotic spindle formation in taxol‐treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers.

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.

Auxin deprivation induces a developmental switch in maize somatic embryogenesis involving redistribution of microtubules and actin filaments from endoplasmic to cortical cytoskeletal arrays.

A developmental switch from non-polar pre-embryogenic units to polarized transition units in maize embryogenic callus is caused by auxin deprivation from the culture medium. This switch is accompanied by cytoskeletal rearrangements in embryogenic cells. An immunofluorescence study revealed prominent endoplasmic microtubules and actin filament meshworks radiating from the nuclear surfaces in pre-embryogenic cells growing on medium supplemented with auxin. On the other hand, parallel-organized cortical microtubules and cortical actin filament networks are inherently associated with polarized embryogenic cells of transition units growing on medium without auxin. These results indicate that fine-tuning of the dynamic equilibrium between endoplasmic and cortical cytoskeletal arrays is important for progress in somatic embryogenesis.

Focal loss of actin bundles causes microtubule redistribution and growth cone turning.

Ît is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.

The Exocyst Complex Associates with Microtubules to Mediate Vesicle Targeting and Neurite Outgrowth

During neuronal development, vesicles are targeted to the growth cone to promote neurite outgrowth and synaptogenesis. The Exocyst complex is an essential macromolecule in the secretory pathway that may play a role in vesicle targeting. Although it has been shown that this complex is enriched in rat brain, the molecular mechanism underlying its function is largely unknown. Here, we report that the Exocyst complex coimmunoprecipitates with microtubules from total rat brain lysate. Additionally, the Exocyst complex subcellular localization changes on neuronal differentiation. In undifferentiated pheochromocytoma (PC12) cells, this complex is associated with microtubules at the microtubule organizing center. However, in differentiated PC12 cells and cultured hippocampal neurons, the Exocyst complex and microtubules extend to the growing neurite and colocalize at the growth cone with synaptotagmin. Inhibition of the NGF-activated MAP kinase pathway blocks the Exocyst complex and microtubule redistribution, abolishing neurite outgrowth and promoting cytosolic accumulation of secretory vesicles. Consistently, the overexpression of Exocyst sec10 subunit mutant blocks neurite outgrowth. These results indicate that the Exocyst complex targets secretory vesicles to specific domains of the plasma membrane through its association with the microtubules, promoting neurite outgrowth.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid.

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.

Depletion of a Microtubule-Associated Motor Protein Induces the Loss of Dendritic Identity

Dendrites are short stout tapering processes that are rich in ribosomes and Golgi elements, whereas axons are long thin processes of uniform diameter that are deficient in these organelles. It has been hypothesized that the unique morphological and compositional features of axons and dendrites result from their distinct patterns of microtubule polarity orientation. The microtubules within axons are uniformly oriented with their plus ends distal to the cell body, whereas microtubules within dendrites are nonuniformly oriented. The minus-end-distal microtubules are thought to arise via their specific transport into dendrites by the motor protein known as CHO1/MKLP1. According to this model, CHO1/MKLP1 transports microtubules with their minus ends leading into dendrites by generating forces against the plus-end-distal microtubules, thus creating drag on the plus-end-distal microtubules. Here we show that depletion of CHO1/MKLP1 from cultured neurons causes a rapid redistribution of microtubules within dendrites such that minus-end-distal microtubules are chased back to the cell body while plus-end-distal microtubules are redistributed forward. The dendrite grows significantly longer and thinner, loses its taper, and acquires a progressively more axon-like organelle composition. These results suggest that the forces generated by CHO1/MKLP1 are necessary for maintaining the minus-end-distal microtubules in the dendrite, for antagonizing the anterograde transport of the plus-end-distal microtubules, and for sustaining a pattern of microtubule organization necessary for the maintenance of dendritic morphology and composition. Thus, we would conclude that dendritic identity is dependent on forces generated by CHO1/MKLP1.

Reorganization and Movement of Microtubules in Axonal Growth Cones and Developing Interstitial Branches

Local changes in microtubule organization and distribution are required for the axon to grow and navigate appropriately; however, little is known about how microtubules (MTs) reorganize during directed axon outgrowth. We have used time-lapse digital imaging of developing cortical neurons microinjected with fluorescently labeled tubulin to follow the movements of individual MTs in two regions of the axon where directed growth occurs: the terminal growth cone and the developing interstitial branch. In both regions, transitions from quiescent to growth states were accompanied by reorganization of MTs from looped or bundled arrays to dispersed arrays and fragmentation of long MTs into short MTs. We also found that long-term redistribution of MTs accompanied the withdrawal of some axonal processes and the growth and stabilization of others. Individual MTs moved independently in both anterograde and retrograde directions to explore developing processes. Their velocities were inversely proportional to their lengths. Our results demonstrate directly that MTs move within axonal growth cones and developing interstitial branches. Our findings also provide the first direct evidence that similar reorganization and movement of individual MTs occur in the two regions of the axon where directed outgrowth occurs. These results suggest a model whereby short exploratory MTs could direct axonal growth cones and interstitial branches toward appropriate locations.

Development of cellular polarity of hamster embryos during compaction.

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.

Development of cellular polarity of hamster embryos during compaction

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.

Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon.

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761-2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617-633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.

Stereological analysis of gravitropism in protonemata of the moss Ceratodon.

Apical cells of dark-grown protonemata of the moss Cerotodon purpureus are negatively gravitropic. Previous light microscopy has shown that reorientation to the horizontal induces amyloplast sedimentation and redistribution of microtubules. To determine whether other components become redistributed laterally or axially, the apical 35 micrometers of both vertical and horizontal apical cells were compared stereologically using transmission electron microscopy. Reorientation to the horizontal changed the longitudinal distributions of tubular ER, Golgi stacks, and vesicles but not cisternal ER, mitochondria, and plastids. Only plastids showed a statistically significant lateral redistribution after horizontal placement. Qualitative examination of the sedimentation zone showed plastids sedimented close to peripherally located ER with vacuoles displaced above plastids. These results argue against a model where differential tip growth results from a redistribution of Golgi stacks or exocytic vesicles.