Talks at the Organelle Structure and Vesicle Formation Minisymposium featured new ways to look at vesicle formation, described molecular links between coat components, and uncovered the unexpected ways that organelles connect with one another.
Vesicle assembly in space and time
Two talks used technological advances in light and electron microscopy to examine the formation of vesicles from the plasma membrane. In yeast, endocytosis proceeds via clathrin-tipped tubular invaginations. Andrea Picco (Kaksonen laboratory, European Molecular Biology Laboratory) used live-cell imaging to reconstruct the dynamic organization of the yeast endocytic machinery, addressing the question of precisely how actin is involved in the budding of endocytic vesicles. Correlating superimposed particle tracks with stage-specific budding profiles from electron microscopy allowed proteins to be mapped with unprecedented spatial and temporal resolution. The actin-binding protein Sla2 was localized to the tip of the invagination. Fluorescence recovery after photobleaching analysis provided evidence that actin polymerizes at the base of the invagination, at the zone where nucleation-promoting factors overlap with Arp2/3. This polymerization could provide force to drive nascent vesicles away from the plasma membrane.
Caveolae—uniform flask-shaped invaginations at the surface of mammalian cells—are coated with caveolin and cavin proteins. How they associate to form caveolar coats has not been clear. Benjamin Nichols (Medical Research Council Laboratory of Molecular Biology) purified stabilized caveolar coat complexes to homogeneity and defined the stoichiometry of the coat complex. Further experiments examined the localization of coat complexes by tagging caveolar proteins with miniSOG (mini Singlet Oxygen Generator), which generates a localized electron-dense stain. Cavins and caveolin were found to be distributed all around caveolae in a regular network.
Linking coat proteins
Other talks examined the regulation of vesicle coats at Golgi and post-Golgi organelles. The COPI coat is thought to act both during transport within the Golgi stack and between the Golgi and the endoplasmic reticulum (ER), raising the possibility that it is regulated in different ways. Scyl1, a catalytically inactive protein kinase implicated in neurodegenerative disease, was previously shown to bind the COPI coat. Jason Hamlin (McPherson laboratory, Montreal Neurological Institute, McGill University) reported that Scyl1 interacts with class II Arfs, the ARF guanine nucleotide exchange factor GBF1, COPI, and membranes, suggesting that it serves as a scaffold for multiple components of the COPI machinery that may be involved in local regulation of coat activity.
The AP-1 adaptor complex incorporates Golgi/endosomal cargo into clathrin-coated vesicles. Elizabeth Conibear (University of British Columbia) described how genome-wide screening was combined with genetic interaction mapping to define new regulators of the AP-1 pathway in yeast. Two of these form a complex that is related to mammalian Fez1/SCOCO and links the adaptin-related protein Laa1 to the AP-1 appendage domain, thereby regulating AP-1’s membrane association.
Organelles get together
Recent data have emphasized that direct contacts between the ER and other organelles play important physiological functions, such as the transfer of lipids between two bilayers or interactions between enzymes and substrates on opposing membranes. However, there is still little molecular detail on how these membrane regions are generated. Francesca Giordano (De Camilli laboratory, Yale University School of Medicine) described a protein family that appears to play a key role in the formation of ER–plasma membrane contact sites and thus has an impact on these processes.
Heidi Hehnly (Doxsey laboratory, University of Massachusetts School of Medicine) reported the intriguing and unexpected observation that recycling endosomes associate specifically with appendages of the mother centriole. The centriolar appendage proteins cenexin and centriolin mediated interactions with GTP-bound Rab11 and the Rab11 GAP, Evi5, respectively. Depletion of centriolin reduced centriole-associated Evi5 and enhanced both Rab11 binding and transferrin recycling, suggesting the Rab11–centrosome connection is important for the function of recycling endosomes.
Footnotes
Molecular Biology of the Cell Volume 24 Page 674
MBoC is pleased to publish this summary of the Minisymposium “Organelle Structure and Vesicle Formation” held at the American Society for Cell Biology 2012 Annual Meeting, San Francisco, CA, December 18, 2012.