David G. Drubin
Department Co-Chair and Ernette Comby Chair in Microbiology Professor of Cell and Developmental Biology*
*And Affiliate, Division of Genetics, Genomics and Development
We use real-time imaging of live cells, genome editing, genetics and biochemistry in human stem cells, in stem-cell derived organoids and in budding yeast to elucidate the molecular mechanisms that underlie highly dynamic actin-mediated membrane trafficking events. We also investigate detailed actin filament assembly mechanisms with purified proteins and in cell extracts to discover new concepts for cytoskeletal force production.
Membrane Trafficking and the Cytoskeleton. Using real-time microscopy and sophisticated analytical tools, coupled with genetics and molecular genetics, we have identified a pathway in budding yeast in which proteins are recruited to endocytic sites in a highly regular, sequential manner. Near the end of this process, a burst of actin assembly facilitates vesicle formation. By studying mutants of over 60 proteins, we have identified several protein modules that provide distinct functions in this pathway. Since actin is among the most highly conserved proteins known, we long believed that the results we obtain from studies in yeast would be directly transferable to more complex eukaryotes including humans. Defects in trafficking events and cytoskeletal proteins are linked to human diseases such as cancer and neuronal degeneration. We are isolating and characterizing mammalian homologues of cytoskeletal proteins that we first identified and characterized in yeast. We are particularly interested in determining the roles of these proteins in endocytosis and cell polarity development. Using genome-editing and stem cells, we are translating some of the experimental advantages of yeast to mammalian cells.
Actin Assembly. Elucidation of the molecular mechanisms used to regulate actin assembly will require a detailed knowledge of how actin subunits assemble into long polymers, and how proteins that bind to monomers and polymers affect assembly dynamics. We have performed a structure-function analysis of actin by mutating residues involved in nucleotide hydrolysis and assaying the effects of these mutations on actin assembly in vitro and in vivo. In complementary studies, genetic, biochemical and structural studies of the low molecular weight (16 kD) actin filament severing protein cofilin and its cofactor, Aip1p, and the actin nucleotide exchange factor, profilin, are being performed to determine how filament turnover and elongation are controlled in vivo. We have also identified and are studying several novel activators of the Arp2/3 complex, which regulates actin nucleation, and we are also studying the role of nucleotide in Arp2/3 function. Recently, we have succeeded in reconstituting a complex actin assembly system on the surface of microbeads incubated in yeast cell extracts, and next aspire to reconstitute complex actin-based trafficking events on supported lipid bilayers. We are also using synthetic biology and biochemistry to elucidate the force generating mechanism for Arp2/3-nucleated actin filament networks and to determine how myosin motors synergize with actin assembly for coordinated force production on membranes.
Type 1 myosins anchor actin assembly to the plasma membrane during clathrin-mediated endocytosis. [Pedersen RTA and Drubin DG (2019) J. Cell Biol. pii: jcb.201810005. doi: 10.1083/jcb.201810005]
Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation. [Dambournet D, Sochaki KA, Cheng AT, Akamatsu M, Taraska JW, Hockemeyer D, Drubin DG (2018) J. Cell Biol. DOI: 10.1083/jcb.201710084]
Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo. [Sun Y, Leong NT, Jiang T, Tangara A, Darzacq X, Drubin DG. (2017) eLife. 2017 Aug 16;6. pii: e29140. doi: 10.7554/eLife.29140]
An engineered minimal WASP-Myosin fusion protein reveals essential functions for endocytosis. [Lewellyn, EB, Pedersen, RTA, Hong, J, Lu, R, Morrison, HM and Drubin, DG. (2015) Dev. Cell 35:281-294.]
Machine-learning-based analysis in genome-edited cells reveals the efficiency of clathrin-mediated endocytosis. [Hong, SH, Cortesio, CL, Drubin, DG. (2015) Cell Reports. http://dx.doi.org/10.1016/j.celrep.2015.08.048]
Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. [Grassart A, Cheng AT, Hong SH, Zhang F, Zenzer N, Feng Y, Briner DM, Davis GD, Malkov D, Drubin DG. (2014) J Cell Biol. Jun 9;205(5):721-35.]
Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms. [Michelot, A, Grassart, A, Okreglak, V, Costanzo, M, Boone, C and Drubin DG. (2013) Dev. Cell 24(2):182-95]
Determinants of endocytic membrane geometry, stability and scission. [Kishimoto T, Sun Y, Buser C, Liu J, Michelot A and Drubin, DG. (2011) Proc. Natl. Acad. Sci USA. 108(44):E979-88]
Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. [Doyon JB, Zeitler B, Cheng J, Cheng AT, Cherone JM, Santiago Y, Lee AH, Vo TD, Doyon Y, Miller JC, Paschon DE, Zhang L, Rebar EJ, Gregory PD, Urnov FD and Drubin DG (2011) Nat. Cell Biol. 13(3):331-7]
A modular design for the clathrin- and actin-mediated endocytosis machinery [Kaksonen, M, Toret, CP and Drubin, DG (2005) Cell 123:305-320]
A pathway for association of receptors, adaptors and actin during endocytic internalization. [Kaksonen, M, Sun, Y and Drubin, DG (2003) Cell 115:475-487]
Photo credit: Mark Hanson at Mark Joseph Studios.
Last Updated 2019-01-30