Microtubule release from the centrosome

Keating, Thomas J.; Peloquin, John; Rodionov, Vladimir; Momcilovic, D.; Borisy, Gary G.

doi:10.1073/pnas.94.10.5078

Cited by 234 publications

(191 citation statements)

References 43 publications

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“…Notably, a specific knockdown of kinesin heavy chain leads to a dramatic reduction of microtubule motions (see SI Text and Movie S13). This further strengthens our hypothesis of rapid motor-induced microtubule motions, in agreement with similar observations in other systems (13)(14)(15)(16)(17)(18).…”

Section: Discussionsupporting

confidence: 81%

The role of microtubule movement in bidirectional organelle transport

Kulić

Brown

Kim³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

135

118

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We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFPtagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks. intracellular transport ͉ molecular motors ͉ kinesin ͉ dynein ͉ cytoskeleton M olecular motor-mediated transport along microtubules is an extensively studied phenomenon in vitro (1-3). Despite significant advances in vitro, understanding how intracellular transport works in vivo still remains one of the big challenges in cell biology. The question of how cellular cargos find their way through the cytoplasm and get targeted to their temporary or final destinations lies at the heart of the problem. One of the major puzzles in this context is the so-called bidirectional organelle transport. The majority of cargos in the cell move in a bidirectional and often remarkably symmetric manner (4, 5). Despite the known kinetic and dynamic asymmetry of the underlying plus-and minus-end directed microtubule motors, the vesicles seem to move with the same rates and run length distributions, and exhibit identical stalling forces, in each direction (4-7). Furthermore, inhibition of transport in one direction typically results in the inhibition of movement in the opposite direction as well (4-11).One straightforward explanation of bidirectional organelle transport rests on the hypothesis (4-11) that a dedicated molecular mechanism couples opposite polarity microtubule motors in vivo. Although this is possible, we suggest an alternative, perhaps complementary hypothesis to the motor coupling hypothesis. Our hypothesis rests on the plausible assumption that a cargo vesicle has multiple motors residing on it and that these couple to several microtubules at a time. The conflicting strains cause them to slide, bend, and buckle, causing effective aperiodic limited-amplitude fluctuations. These fluctuations modify the motion of vesicles in an additive manner, contributing to the phenomenology of bidirectional organelle transport. We describe unusual observations coming from high-resolution traces of single peroxisomes, in particular, the unusual mean-square displacement (MSD) behavior and large velocity crosscorrelation observed between peroxisomes. We then present results from simultaneous two-color imaging of peroxisome...

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Section: Discussionsupporting

confidence: 81%

The role of microtubule movement in bidirectional organelle transport

Kulić

Brown

Kim³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

135

118

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“…This means that our in vivo observations were likely not to result from the severing and release of centrosomal MTs (Baas and Joshi, 1992;Yu et al, 1993;Keating et al, 1997;Vorobjev et al, 1997) or even spontaneous cytoplasmic assembly, followed by treadmilling or migration en bloc Vorobjev et al, 1997;Yvon and Wadsworth, 1997;Tucker et al, 1998) until they were captured and stabilized by scattered Golgi elements. Because the reconstitution of mini-Golgi stacks at ER exit sites actively participates in the dispersion of the Golgi complex after nocodazole-mediated MT depolymerization (Cole et al, 1996;Storrie et al, 1998), the ER would also have been a likely candidate for stimulating noncentrosomal MT assembly.…”

Section: Discussionmentioning

confidence: 99%

The Golgi Complex Is a Microtubule-organizing Organelle

Chabin-Brion

Marceiller

Perez

et al. 2001

MBoC

277

245

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We show that the Golgi complex can directly stimulate microtubule nucleation in vivo and in vitro and thus behaves as a potent microtubule-organizing organelle in interphase cells. With the use of nocodazole wash-out experiments in hepatic cells, we found that the occurrence of noncentrosomal, early stabilized microtubules is highly correlated with the subcellular localization of Golgi membranes. With the use of in vitro reconstituted microtubule assembly systems with or without cytosol, we also found that, in contrast to centrosomally attached microtubules, the distal ends of Golgi-attached microtubules are remotely stabilized in a way that requires additional cytosolic component (s). Finally, we demonstrate that Golgi-based microtubule nucleation is direct and involves a subset of ␥-tubulin bound to the cytoplasmic face of the organelle. INTRODUCTIONIn interphase cells, the microtubule (MT) network plays a major role in membrane dynamics and organelle localization. In this context, the interaction between the Golgi complex and the MT network has been extensively studied. The Golgi apparatus colocalizes with the minus ends of MTs, which are usually associated with the centrosome (for review see Kreis et al., 1997;Thyberg and Moskalewski, 1999). This localization actually results from an equilibrium between two contradictory movements that have been unraveled with the use of MT-depolymerizing drugs and probably occur on MT subpopulations with different dynamics. The dispersal of Golgi elements occurs along stable MTs after depolymerization of the most labile MT population (Minin, 1997), whereas their reclustering involves newly assembled MTs (Ho et al., 1989). These contradictory movements of Golgi elements are mediated by distinct molecular motors. Consistent with earlier findings by Feiguin et al. (1994), who found that the expression of kinesin antisense oligonucleotide rendered the Golgi apparatus more compact, microinjection of antikinesin antibodies inhibited Golgi dispersion along stable, nocodazole-resistant MTs (Minin, 1997). Conversely, the central localization of the Golgi apparatus involves cytoplasmic dynein (Corthésy-Theulaz et al., 1992;Fath et al., 1994;Burkhardt et al., 1997;Harada et al., 1998). It is also worth noting that, in addition to the kinesin-mediated disruption of the central Golgi complex during MT depolymerization, the dispersion of Golgi elements also relies on the reconstitution of mini Golgi stacks at endoplasmic reticulum (ER) exit sites (Cole et al., 1996;Storrie et al., 1998). Such data on the scattering and the reclustering of the Golgi complex have led to the view that Golgi membranes undergo an intimate relationship with MTs and especially with a population of nocodazole-resistant, stable MTs. Stable MTs are characterized by the occurrence of posttranslationally modified tubulin-especially detyrosinated and acetylated tubulin (for review, see MacRae, 1997), which is thought to accumulate in stable MTs because of their longer half-lives, but does not influence MT stability i...

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“…T ubulin was prepared from bovine brain (Hyman et al, 1991) and labeled with tetramethyl-rhodamine (TMR) as described previously (Keating et al, 1997). In some experiments, rhodamine-tubulin was purchased (C ytoskeleton Inc.).…”

Section: Methodsmentioning

confidence: 99%

Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments

Dent¹,

2001

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Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence timelapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth. Key words: microtubule; actin filament; growth cone; collateral axon branching; cortical development; fluorescence timelapse imagingAxons are guided in new directions by reorientation of their growth cones as well as extension of collateral branches (O'Leary et al., 1990). We have shown previously (Szebenyi et al., 1998) that cortical axon branching occurs in vitro through changes in growth cone morphologies and behaviors. Growth cones at the tips of rapidly extending cortical axons are typically small and highly motile. However, in preparation for branching, growth cones pause for many hours, greatly enlarge, and maintain motility without forward advance. Subsequently, a new growth cone develops from the tip of the large paused growth cone and forms the new leading axon. Remnants of the large paused growth cone remain behind on the axon shaft as filopodial and lamellar expansions that subsequently give rise to interstitial axon collaterals. In living cortical slices (Halloran and Kalil, 1994), similar growth cone pausing behaviors were observed in the corpus callosum in regions where collateral axon branches develop and extend toward cortical targets, suggesting that growth cone pausing is closely related to branching mechanisms in vivo.Changes in the direction of axon outgrowth depend on reorganization of the microtubule and actin cytoskeleton (Lin and

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Microtubule release from the centrosome

Cited by 234 publications

References 43 publications

The role of microtubule movement in bidirectional organelle transport

The role of microtubule movement in bidirectional organelle transport

The Golgi Complex Is a Microtubule-organizing Organelle

Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments

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