Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.
Yidi Sun‘s new paper is out now as an electronic publication ahead of print in the Journal of Cell Science. Congratulations to Yidi on her great work! The abstract is below. The PDF can be downloaded from JCS here.
Anionic phospholipids PI(4,5)P(2) and phosphatidylserine (PS) are enriched in the cytosolic leaflet of the plasma membrane where endocytic sites form. In this study, we investigated the roles of PI(4,5)P(2) and PS in clathrin-mediated endocytosis (CME) site initiation and vesicle formation in Saccharomyces cerevisiae. Live-cell imaging of endocytic protein dynamics in an mss4(ts) mutant, which has severely reduced PI(4,5)P(2) levels, revealed that PI(4,5)P(2) is required for endocytic membrane invagination but is less important for endocytic site initiation. We also demonstrated that in various deletion mutants of genes encoding components of the Rcy1-Ypt31/32 GTPase pathway, endocytic proteins dynamically assemble not only on the plasma membrane but also on intracellular membrane compartments, which are likely derived from early endosomes. In rcy1Δ cells, fluorescent biosensors indicated that PI(4,5)P(2) only localized to the plasma membrane while PS localized to both the plasma membrane and intracellular membranes. Furthermore, we found that polarized endocytic patch establishment is defective in the PS-deficient cho1Δ mutant. We propose that PS is important for directing endocytic proteins to the plasma membrane and that PI(4,5)P(2) is required to facilitate endocytic membrane invagination.
“The difference between taking snapshots of the process and watching a movie is just night and day,” says David Drubin, Ph.D., professor of cell and developmental biology at the University of California, Berkeley, whose lab uses fluorescence to understand the intricate details underlying clathrin-mediated endocytosis.
Researchers in David Drubin’s lab at the University of California, Berkeley genetically engineered a human cell line to express endogenous levels of RFP-tagged clathrin light chain A (red) and GFP-tagged dynamin 2 (green) for studying clathrin-mediated endocytosis. The above 3D kymograph of the cell surface, with the time dimension in the z-axis, shows the full lifetime of hundreds of clathrin patches on the membrane, which terminate upon recruitment of dynamin. [Aaron T. Cheng]
The May 2012 edition of Biowire, a publication of Sigma-Aldrich, includes an interview with David Drubin about the projects in our lab looking at clathrin-mediated endocytosis (CME) in mammalian cells using zing finger nuclease (ZFN) technology to undertake targeted genome modification. Traditionally, CME has been studied in cells in which fluorescently-tagged components of endocytic machinery are overexpressed using exogenous constructs. Data obtained in many labs using these methods suggested that CME was highly variable. Using ZFN technology, in collaboration with Sangamo Biosciences, our lab recently showed that CME is robust and efficient in mammalian cells. The new results highlight the technical advantages of tagging genes at their endogenous loci, an approach that has been historically limited to genetically tractable organisms, such as the Drubin/Barnes Lab favorite Saccharomycescerevisiae (budding yeast). Emerging technologies, such as ZFNs and TALENs, however, are now making this sort of precise genomic manipulation possible in animal cells, including human cells, giving us new and powerful ways of studying cellular biology.
Cellular processes should be studied as close to their natural states as possible. I suspect that, as we see more uses of zinc finger nucleases [for tagging endogenous genes], people will find that they have been inadvertently perturbing the processes that they have been studying.