Biomedical subjects

Roberto Weigert

Publications and source records attributed to Roberto Weigert.

6 recordsLinked to original sources

PubMed2005

Multiple activities for Arf1 at the Golgi complex.

The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.

ADP-Ribosylation Factor 1↗

PubMed2005

Fluorescent microscopy-based assays to study the role of Rab22a in clathrin-independent endocytosis.

The endocytic and trafficking route followed by proteins that enter cells independently of clathrin is not well understood. In HeLa cells, a distinct endocytic and recycling pathway is followed for such clathrin-independent cargo proteins, and we have characterized the role of Rab proteins in this process. Here we describe cell-based flourescence assays to examine the effects of expression and depletion of Rab22 on endosomal morphology and endocytic recycling.

Base Sequence↗

PubMed2004

Rab22a regulates the recycling of membrane proteins internalized independently of clathrin.

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.

Animals↗

PubMed2004

Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements.

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.

ADP-Ribosylation Factor 6↗

PubMed2003

A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

In mitosis, chromosome, cytoskeleton, and organelle dynamics must be coordinated for successful cell division. Here, we present evidence for a role for Arf1, a small GTPase associated with the Golgi apparatus, in the orchestration of mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We show that early in mitosis Arf1 becomes inactive and dissociates from Golgi membranes. This is followed by the dispersal of numerous Arf1-dependent peripheral Golgi proteins and subsequent Golgi disassembly. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of its GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. These cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinetic furrow ingression. These findings suggest that mitotic Golgi disassembly depends on Arf1 inactivation and is used by the cell to disperse numerous peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis.

ADP-Ribosylation Factor 1↗

PubMed2003

Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides.

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.

ADP-Ribosylation Factor 6↗