Rab3A and Rab3D Control the Total Granule Number and the Fraction of Granules Docked at the Plasma Membrane in PC12 Cells

Martelli, Alberto M.; Baldini, Giovanna; Tabellini, Giovanna; Koticha, Darshan; Bareggi, Renato

doi:10.1111/j.1600-0854.2000.11207.x

Cited by 47 publications

(39 citation statements)

References 42 publications

(78 reference statements)

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“…In light of a recent study on PC12 cells by Martelli and colleagues our data strongly support this idea. They showed that the overexpression of a rab3a mutant protein deficient in GTP hydrolysis led to a decrease in the total number of SGs in the vicinity of the PM (Martelli et al, 2000). This is in agreement with our finding of a reduction in cortical localisation in the presence of the tail fragment of myosin Va.…”

Section: Existence and Recruitment Of Motor Protein Complexessupporting

confidence: 83%

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells

Rudolf

Kögel

Kuznetsov

et al. 2003

Journal of Cell Science

120

131

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Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the transGolgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells. Movie available online

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Section: Existence and Recruitment Of Motor Protein Complexessupporting

confidence: 83%

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells

Rudolf

Kögel

Kuznetsov

et al. 2003

Journal of Cell Science

120

131

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“…The observation that Ca 2ϩ acts in the final stage of exocytosis, which was also identified in the Rab3A knock-out as the point where the function of Rab3A becomes apparent, reconciles the knock-out data and the PC12 cell transfection data. This observation also suggests the reason that a decrease in the number of total and docked vesicles was observed in transfected PC12 cells expressing Rab3A and 3D (20). Although viewed together, the results reported here and previously argue for a general role of Rab3 proteins in the final stage of exocytosis, the mechanism involved is still unclear.…”

Section: Discussionmentioning

confidence: 47%

“…In this regard, Rab3 proteins are similar to Rab11b, which we recently found to be the only Rab protein tested among multiple such proteins that inhibited exocytosis. 2 Overall, the available data reported in this and other studies (12,14,20) indicate that Rab3A, Rab3B, Rab3C, and Rab3D are functionally comparable based on similar localizations, a high degree of sequence homology, and equivalent inhibition of exocytosis in transfected PC12 cells. Clearly there are major differences in the cells and tissues that express the different Rab3 isoforms and possibly also in the functions of Rab3 proteins, because the biogenesis and fusion of secretory vesicles in exocrine glands containing Rab3D exhibit quite different properties from the biogenesis and fusion of endocrine and neuronal secretory vesicles.…”

Section: Discussionmentioning

confidence: 77%

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Localization Versus Function of Rab3 Proteins

Schlüter

Khvotchev

Jahn

et al. 2002

Journal of Biological Chemistry

150

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Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca 2؉-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca 2؉ -triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lü thi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca 2؉ -independent exocytosis that correlated with the inhibition of regulated Ca 2؉ -triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knockout studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.

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“…Synaptic vesicles fail to cluster near release sites in C. elegans and mouse Rab3 mutants (Nonet et al, 1997;Leenders et al, 2001), and overexpression of Rab3 in PC12 cells increases the number of docked granules (Martelli et al, 2000). The yeast Rab3 homolog Ypt7p interacts with a complex containing the UNC-18 homolog Vps33p, and another yeast Rab3 homolog, Ypt1p, interacts genetically with the UNC-18 homolog Sly1p (certain Sly1 mutations bypass loss of Ypt1p) (Ossig et al, 1991).…”

Section: Dockingmentioning

confidence: 99%