Dynactin Is Required for Microtubule Anchoring at Centrosomes

Quintyne, Nicholas J.; Gill, Steven R.; Eckley, D. Mark; Crego, C.L.; Compton, Duane A.; Schroer, Trina A.

doi:10.1083/jcb.147.2.321

Cited by 324 publications

(456 citation statements)

References 52 publications

(114 reference statements)

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“…Dynamitin may stabilize the dynein-dynactin interaction by binding dynein directly. We find (Quintyne et al, 1999) that overexpression of different dynactin subunits can have a variety of effects, including Golgi complex and endosome dispersion, disorganization of the interphase microtubule array, and disruption of dynactin structure. Dynamitin induces all these effects, whereas other dynactin subunits such as p150 Glued disrupt Golgi structure and microtubule organization without affecting dynactin integrity.…”

Section: Discussionmentioning

confidence: 89%

“…The dynamitin cDNA insert was subcloned into two cytomegalovirus (CMV)-based expression vectors: pGW1-CMV (Quintyne et al, 1999) and pCB6 containing an upstream HA tag (Balda et al, 1996). To facilitate subcloning, the first 108 bp of the dynamitin cDNA was amplified by PCR using an AlfII site introduced at the dynamitin initiator codon and a unique, downstream SacI site.…”

Section: Expression Constructsmentioning

confidence: 99%

“…The insert was verified by sequencing and cloned into pGW1-CMV. A gt10 insert encoding full-length chicken p150 was cloned as described (Quintyne et al, 1999). Plasmid pSG5-myc-CLIP-170 wild type (wt) contained the EcoRI insert from pGEM-myc-CLIP (Pierre et al, 1994) subcloned into pSG5; pSG5-myc-CLIP⌬1240 was generated by truncating pSG5-myc-CLIP-170 wt using XhoI and BamHI followed by blunting and religation (both were gifts from J. Rickard, University of Geneva).…”

Section: Expression Constructsmentioning

confidence: 99%

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Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Valetti

Wetzel

Schrader

et al. 1999

MBoC

256

252

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The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

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Section: Discussionmentioning

confidence: 89%

Section: Expression Constructsmentioning

confidence: 99%

Section: Expression Constructsmentioning

confidence: 99%

See 1 more Smart Citation

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Valetti

Wetzel

Schrader

et al. 1999

MBoC

256

252

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“…At the level of the centrosome, a sequential model has emerged in which MTs are first nucleated on ␥-tubulin-containing complexes and then released and anchored on distinct sites (Quintyne et al, 1999;Mogensen et al, 2000). A similar model might also apply for the Golgi complex in which the Golgi-associated MAP GMAP-210 that was shown to bind both the minus end of MTs and CGN membranes (Infante et al, 1999) would be a good docking candidate.…”

Section: Discussionmentioning

confidence: 99%

The Golgi Complex Is a Microtubule-organizing Organelle

Chabin-Brion

Marceiller

Perez

et al. 2001

MBoC

275

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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“…Because dynactin ensures Golgi complex integrity (Quintyne et al, 1999) and its intracellular movements, we decided to study the influence of LOSK inhibition on Golgi apparatus.…”

Section: The Inhibition Of Losk Activity Does Not Influencementioning

confidence: 99%

Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

Burakov

Zhapparova

Kovalenko

et al. 2008

MBoC

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Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-⌬T or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-⌬T have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types. INTRODUCTIONThe radial array of microtubules is typical for many mammalian cells. It organizes bidirectional organelle transport in the cytoplasm in the endocytotic and exocytotic direction. It is also required for the regulation of interaction of microtubule plus ends with cell periphery. Both functions are important for cell polarization, movement, and signal transduction (Hyman and Karsenti, 1996;Dujardin et al., 2003;Morrison, 2007). The degree of radiality of microtubules varies in different types of cells. For instance, in fish melanophores the system of microtubules is perfectly radial (Schliwa et al., 1978), whereas in myotubes it is unclear (Tassin et al., 1985;Musa et al., 2003). Moreover, among fibroblast-like cultured mammalian cells some (green monkey kidney Vero or Chinese hamster ovary CHO-K1) possess a distinct radial microtubule array (Bre et al., 1987), whereas others (human cervical carcinoma HeLa or mouse fibroblasts NIH 3T3) have rather chaotic microtubule arrangements (Bulinski and Borisy, 1980). Regardless of the tissue origin of cells, microtubules become more radial when cultured cells are sparse and less radial in confluent cultures. It seems that the status of microtubule organization is regulated by signal transduction pathways and depends on cell differentiation.The focused microtubule arrays can also be formed in acentrosomal cell fragments as a result of interactions between microtubules and membrane vesicles, covered with motor proteins (Rodionov and Borisy, 1997;Malikov et al., 2005). In acentrosomal fragments of fish melanocytes microtubules are chaotic when pigment granules are dispersed. After the addition of adrenaline or other activation of melanosome aggregation microtubules assemble in a radial array. Thus, in this case...

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Dynactin Is Required for Microtubule Anchoring at Centrosomes

Cited by 324 publications

References 52 publications

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

The Golgi Complex Is a Microtubule-organizing Organelle

Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

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