Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells.

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature.

Caveolin regulates endocytosis of the muscle repair protein, dysferlin.

Dysferlin and Caveolin-3 are plasma membrane proteins associated with muscular dystrophy. Patients with mutations in the CAV3 gene show dysferlin mislocalization in muscle cells. By utilizing caveolin-null cells, expression of caveolin mutants, and different mutants of dysferlin, we have dissected the site of action of caveolin with respect to dysferlin trafficking pathways.

Functional assay of ARNO and ARF6 in neurite elongation and branching.

During development of the nervous system, neurite outgrowth is necessary for the formation of connections between nerve cells. Neurons are highly polarized cells that send out distinct processes, axons, and dendrites; however, the molecular regulation of the differential growth of these processes remains incompletely understood. Primary cultures of rat hippocampal neurons have been used to study many aspects of neuronal cell biology, including neurite extension, establishment of polarity, biogenesis of synapses, and membrane trafficking.

Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3.

Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts.

ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha.

In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking.

Regulation of dendritic development by the ARF exchange factor ARNO.

Here we analyzed the role of ARF6, a member of the ADP-ribosylation factor (ARF) family of small GTPases, in dendritic arbor development in rat hippocampal neurons in culture. Overexpression of the inactive form of the GTP exchange factor ARNO (ARF nucleotide binding site opener) or inactive ARF6 enhanced dendritic branching, whereas coexpression of either Rac1 (a member of the Rho family of small GTPases known to control dendritic dynamics and growth) or active ARF6 with inactive ARNO eliminated the enhanced branching effect. These results indicate that the ARF family of small GTPases contributes to the regulation of dendritic branching, and that ARF6 activation turns on two independent pathways that suppress dendritic branching in vivo: one through Rac1 and the other through ARF6.