Biomedical subjects

Ramiro H Massol

Publications and source records attributed to Ramiro H Massol.

5 recordsLinked to original sources

PubMed2006

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Animals↗

PubMed2005

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking.

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.

ADP-Ribosylation Factor 6↗

PubMed2004

Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways.

Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.

ADP-Ribosylation Factor 6↗

PubMed2003

HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity.

HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.

ADP-Ribosylation Factor 6↗

PubMed2003

Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission.

Mitochondria undergo cycles of fusion and fission crucial for organelle homeostasis. Fission is regulated partially by recruitment of the large GTPase Dnm1p to the outer mitochondrial membrane. Using three-dimensional time-lapse fluorescence imaging of Saccharomyces cerevisiae cells, we found that Dnm1p-EGFP appears and disappears at "hot spots" along mitochondrial tubes. It forms patches that convert rapidly into different shapes regardless of whether mitochondrial fission ensues or not. Moreover, the thickness of the mitochondrial matrix displays frequent temporal fluctuations apparently unrelated to fission or to recruitment of Dnm1p-EGFP. These results suggest that mitochondrial fission requires coordination of at least two distinct processes.

GTP Phosphohydrolases↗