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Biomedical subjects

Christina Wasmeier

Publications and source records attributed to Christina Wasmeier.

6 recordsLinked to original sources
PubMed2005

Rab8 regulates the actin-based movement of melanosomes.

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes.

Actins↗
PubMed2005

An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin.

Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.

Amino Acid Motifs↗
PubMed2004

Multiple regions contribute to membrane targeting of Rab GTPases.

Small GTPases of the Rab family are key regulators of membrane trafficking. Each Rab shows a characteristic subcellular distribution, and may serve as an important determinant of organelle identity. The molecular mechanisms responsible for targeting Rabs to specific intracellular compartments, however, remain poorly understood. The divergent C-terminal hypervariable region was postulated to contain Rab targeting information. We generated a series of hybrid Rab proteins by exchanging the hypervariable domains of Rab1a, Rab2a, Rab5a, Rab7 and Rab27a, and analysed their subcellular localisations. We found that the various hybrid proteins retained their targeting to the parent organelle and were functionally active. We conclude that the hypervariable region does not contain a general Rab targeting signal. Furthermore, we identified other regions within the RabF and RabSF motifs that are required for specific targeting of Rab27a to secretory granules or melanosomes, and Rab5a to endosomes. We observed only partial overlap between targeting-determining regions in the Rab proteins examined, suggesting that Rab recruitment may be complex and at least partially Rab-specific. Mutations in these targeting-determining regions induced localisation to the ER, an observation that further strengthens our previous finding that ER/Golgi membranes serve as the default location for Rabs that have lost targeting information.

Animals↗
PubMed2004

Controlling the location and activation of Rab GTPases.

The remarkable degree of specificity with which Rab GTPases recognise distinct subsets of intracellular membranes forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the right membrane at the right time. The molecular mechanisms controlling Rab localisation, however, have proved tricky issues to address. It is becoming increasingly apparent that multiple factors contribute to the specificity of Rab localisation and the close coordination of membrane targeting with Rab activation. With important new insights into the mode of action of the general Rab regulators REP and RabGDI, as well as the demonstration that novel factors such as Yip3/Pra1 act as GDI displacement factors and that signals within Rab proteins contribute to targeting specificity, a better understanding of the concepts governing Rab recruitment and function is now beginning to emerge. The diversity of cellular processes regulated by Rab family members is made possible, not only by the wide range of effectors they recruit, but also by the different mechanisms regulating their own targeting and activation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules.

Phogrin, a transmembrane glycoprotein of neuroendocrine cells, is localized to dense-core secretory granules. We have investigated the subcellular targeting of phogrin by analyzing the sorting of a series of deletion mutants to the regulated pathway of secretion in AtT20 cells. The lumenal domain as a soluble protein was efficiently routed to granules, based on a combination of morphological analysis and secretion studies. Sorting was not dependent on a candidate targeting signal consisting of an N-terminal conserved cysteine-rich motif. Both the pro-region and the lumenal domain of mature, post-translationally processed phogrin independently reached the granule, although the pro-region was sorted more efficiently. Once within the regulated secretory pathway, all phogrin lumenal domain proteins were stored in functional granules for extended periods of time. Thus, phogrin possesses several domains contributing to its targeting to the secretory granule. Our findings support a model of granule biogenesis where proteins are sorted on the basis of their biochemical properties rather than via signal-dependent binding to a targeting receptor. Sorting of integral membrane proteins mediated by the lumenal domain may ensure that functionally important transmembrane molecules are included in the forming granule.

Amino Acid Sequence↗