Non‐centrosomal microtubule‐organising centres in cold‐treated cultured <i>Drosophila</i> cells

Cottam, Deborah M.; Tucker, John B.; Rogers-Bald, Margaret M.; Mackie, John B.; Macintyre, John; Scarborough, Julie; Ohkura, Hiroyuki; Milner, Martin J.

doi:10.1002/cm.20103

Cited by 22 publications

(30 citation statements)

References 44 publications

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“…At 10 min, MT foci were more numerous and assembled into an extensive interconnecting MT network throughout the cell ( Figure 6A, 10 min). By 15 min, S2 cells displayed a normal interphase array, having dispersed the MT foci that formed in the earlier time points ( Figure 6A, 15 min).Previous MT regrowth experiments using cultured fly cells described the appearance of unique MT foci similar to what we observed (Colombié et al, 2006;Cottam et al, 2006). Although individual MTs and foci form independently of interphase centrioles in S2 cells (our findings, Figure 6A), ␥-tubulin colocalizes with MT foci that assemble in cold- …”

supporting

confidence: 79%

A Multicomponent Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in InterphaseDrosophilaCells

Rogers¹,

Rusan²,

Peifer

et al. 2008

MBoC

131

133

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In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both ␥-tubulins. However, ␥-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted ␥-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila. INTRODUCTIONThe microtubule (MT) cytoskeleton is essential for organizing the cytoplasm, polarity establishment, cell motility, morphogenesis, and cell division. Polarized arrays of MTs are nucleated by the centrosome, an organelle consisting of a pair of "mother-daughter" centrioles that recruit and organize a surrounding matrix of pericentriolar material (PCM;Bornens, 2002). Proteomic analyses of centrosomes identified several hundred different proteins, some of which are unique to centrioles (e.g., the structural subunits SAS-4, SAS-6, and SPD-2) or the PCM (e.g., pericentrin; Andersen et al., 2003; Keller et al., 2005;Leidel and Gonczy, 2005a). Within the PCM, pericentrin and other coiled-coil proteins assemble into a scaffold that docks ␥-tubulin ring complexes, which nucleate MT growth (Moritz and Agard, 2001). By manipulating centrosome number and position, cells exert precise control over the MT arrays needed for a variety of critical MT-dependent processes.Centrosomes adhere to a cycle of duplication and function that is intimately coupled to the cell cycle (Tsou and Stearns, 2006). Centrosomes act as dominant MT-organizing and nucleating centers (MTOCs;Schiebel, 2000); however, this activity is modulated in a cell cycle-dependent manner. During interphase, cells possess two paired centrioles that form a single centrosome and organize the interphase MT array. Before mitotic entry the centrosome duplicates once; the two centrosomes then separate from one ano...

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supporting

confidence: 79%

A Multicomponent Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in InterphaseDrosophilaCells

Rogers¹,

Rusan²,

Peifer

et al. 2008

MBoC

131

133

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“…The reorganization of the actin and MT cytoskeletons during myogenesis has long been observed in cell culture as individual myoblasts fuse into multinucleated myotubes (Bugnard et al, 2005;Cottam et al, 2006;Musa et al, 2003;Pizon et al, 2005;Tassin et al, 1985). Although the actin cytoskeleton has been shown to be…”

Section: Discussionmentioning

confidence: 99%

“…S3A,B in the supplementary material). γ-tubulin requires RacGAP for its localization to the nuclear periphery Studies in cultured myotubes have shown that γ-tubulin, a minusend anchoring protein essential for MT nucleation at the centrosome (Gunawardane et al, 2000;Stearns et al, 1991), may also be able to promote nucleation from non-centrosomal sites along the nuclear membrane and throughout the cytoplasm (Bugnard et al, 2005;Cottam et al, 2006;Musa et al, 2003). Based upon the previously defined role for RacGAP in MT organization during cell division, as well as our current findings that MT polarity in myotubes is altered in RacGAP and pav mutants (Fig.…”

Section: Research Articlementioning

confidence: 99%

“…These processes are well conserved and are observed in cultured myocytes from diverse organisms including human, mouse and Drosophila (Bugnard et al, 2005;Cottam et al, 2006;Musa et al, 2003;Pizon et al, 2005;Tassin et al, 1985). However, little is known about the molecular mechanisms that drive these morphological changes during muscle development in vivo.…”

Section: Introductionmentioning

confidence: 99%

“…This non-centrosomal MT array is based at the nuclear periphery and extends out in the longitudinal axis of the myotube (Bugnard et al, 2005;Musa et al, 2003;Pizon et al, 2005;Tassin et al, 1985) (reviewed by Bartolini and Gundersen, 2006). In addition, studies in cultured myotubes show that γ-tubulin, a minus-end anchoring protein essential for MT nucleation at the centrosome (Gunawardane et al, 2000;Stearns et al, 1991), may also be able to promote nucleation from non-centrosomal sites along the nuclear membrane and throughout the cytoplasm (Bugnard et al, 2005;Cottam et al, 2006;Musa et al, 2003). Although the reorganization of the MT cytoskeleton during muscle differentiation has been well described in cell culture, little is known about the role of this reorganization during muscle development.…”

Section: Introductionmentioning

confidence: 99%

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RacGAP50C directs perinuclear γ-tubulin localization to organize the uniform microtubule array required forDrosophilamyotube extension

Guerin

Kramer

2009

Development

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The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. We have identified a novel postmitotic function for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. We show that the localization of the MT nucleator γ-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and that this change in localization depends on RacGAP50C. RacGAP50C and γ-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants γ-tubulin remains dispersed throughout the cytoplasm. Furthermore, we show that the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute γ-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process.

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The Drosophila CLASP homologue, Mast/Orbit regulates the dynamic behaviour of interphase microtubules by promoting the pause state

Sousa

Reis

Sampaio

et al. 2007

Cell Motil. Cytoskeleton

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An important group of microtubule associated proteins are the plus-end tracking proteins which includes the Mast/Orbit/CLASPs family amongst others. Several of these proteins have important functions during interphase and mitosis in the modulation of the dynamic properties of microtubules, however, the precise mechanism remains to be elucidated. To investigate the role of Mast in the regulation of microtubule behaviour during interphase, we used RNAi in Drosophila S2 culture cells stably expressing GFP-alpha-tubulin and followed the behaviour of microtubules in vivo. Mast depleted cells show a significant reduction of microtubule density and an abnormal interphase microtubule array that rarely reaches the cell cortex. Analysis of the dynamic parameters revealed that in the absence of Mast, microtubules are highly dynamic, constantly growing or shrinking. These alterations are characterized by a severe reduction in the transition frequencies to and from the pause state. Moreover, analysis of de novo microtubule polymerization after cold treatment showed that Mast is not required for nucleation since Mast depleted cells nucleate microtubules soon after return to normal temperature. Taken together these results suggest that Mast plays an essential role in reducing the dynamic behaviour of microtubules by specifically promoting the pause state.

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Non‐centrosomal microtubule‐organising centres in cold‐treated cultured Drosophila cells

Cited by 22 publications

References 44 publications

A Multicomponent Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in InterphaseDrosophilaCells

A Multicomponent Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in InterphaseDrosophilaCells

RacGAP50C directs perinuclear γ-tubulin localization to organize the uniform microtubule array required forDrosophilamyotube extension

The Drosophila CLASP homologue, Mast/Orbit regulates the dynamic behaviour of interphase microtubules by promoting the pause state

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