We are focused on two key questions in cell biology. First,
how is the actin cytoskeleton regulated to produce mechanical
force and polarity underlying different cellular processes?
These include cell motility, endocytosis, vesicle and organelle
transport, and cytokinesis. More specifically, we are studying
how the diverse activities of numerous actin-associated
proteins are coordinated in cells to control dynamic rearrangements
of actin structures (figure 1). Second, how do the basic
cytoskeletal elements found in most eukaryotic cells (microtubules,
actin, septins, and intermediate filaments) cooperate functionally
during these same cellular processes? We are using a powerful
combination of genetic, biochemical and cell biological
approaches to address these questions in budding yeast (Saccharomyces
cerevisiae), where the core components of the cytoskeleton
are highly conserved with mammals.
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A critical breakthrough in our work was our development
of an assay to study actin assembly in yeast extracts (manuscript
in preparation). For over a decade, it has been known that
the cytoskeleton disassembles rapidly upon cell lysis in
yeast, and it has been difficult to re-stimulate actin assembly
in lysates. We now have developed a procedure to trigger
the re-assembly of > 90% of the actin into organized filamentous
structures. This method provides an unprecedented opportunity
to compare actin assembly in mutant and wild type extracts.
Further, this process can be studied in real time by spiking
reactions with rhodamine-actin and monitoring actin assembly
by fluorescence microscopy.
The actin structures reconstituted in extracts can be isolated
rapidly. This allows us to purify virtually the entire actin
cytoskeleton in a single step. Using mass spectrometry,
we have identified all of the components of this mixture,
defined the relative abundance of each actin-associated
protein in cells, and identified novel functional links
between actin and other cellular processes. Further, by
fractionating the isolated actin mixtures using gel filtration
chromatography, we have identified new activities and functional
interactions among components. For example, we showed that
the endocytic adapter protein Abp1p is a novel activator
of Arp2/3 complex, which plays a central role in promoting
actin assembly in cells (Goode et al., 2001).
Because yeast is highly amenable to genetic studies, we
can introduce mutations into any actin -associated factor
and compare its mutant activities to wild type protein.
We now are using this approach to dissect in vivo functions
and biochemical activities of each sub-unit of the Arp2/3
complex.
A long-range goal of our lab is to develop assays in extracts
to study functional interactions among the different cytoskeletal
systems. Genetic analyses have shown that cytoskeletal networks
cooperate functionally during many different cellular processes
(reviewed in Goode et al., 2000), including nuclear migration
(actin, microtubules, and intermediate filaments), organelle
inheritance (actin and intermediate filaments), and cytokinesis
(actin and septins). Our goal is to reconstitute these interactions
in cell-free extracts and identify the key factors regulating
the interactions.
Selected Publications
Gandhi M, Goode BL, Chan CS. (2006) Four novel suppressors
of gic1 gic2 and their roles in cytokinesis and polarized
cell growth in Saccharomyces cerevisiae.Genetics. 2006 Oct;174(2):665-78.
Epub 2006 Jul 2. [abstract]
Moseley JB, Goode BL. (2006) The yeast actin cytoskeleton:
from cellular function to biochemical mechanism. Microbiol
Mol Biol Rev. 2006 Sep;70(3):605-45. Review. [abstract]
Okada K, Ravi H, Smith EM, Goode BL. (2006) Aip1 and cofilin
promote rapid turnover of yeast actin patches and cables:
a coordinated mechanism for severing and capping filaments.Mol
Biol Cell. 2006 Jul;17(7):2855-68. Epub 2006 Apr 12. [abstract]
Moseley JB, Okada K, Balcer HI, Kovar DR, Pollard TD, Goode
BL. (2006) Twinfilin is an actin-filament-severing protein
and promotes rapid turnover of actin structures in vivo.
J Cell Sci. 2006 Apr 15;119(Pt 8):1547-57. Epub 2006 Mar
28. [abstract]
Moseley JB, Maiti S, Goode BL. (2006) Formin proteins:
purification and measurement of effects on actin assembly.
Methods Enzymol. 2006;406:215-34. [abstract]
D'Agostino JL, Goode BL. (2005) Dissection of Arp2/3 complex
actin nucleation mechanism and distinct roles for its nucleation-promoting
factors in Saccharomyces cerevisiae. Genetics. 2005 Sep;171(1):35-47.
[abstract]
Moseley JB, Goode BL. (2005) Differential activities and
regulation of Saccharomyces cerevisiae formin proteins Bni1
and Bnr1 by Bud6. J Biol Chem. 2005 Jul 29;280(30):28023-33.
[abstract]
Quintero-Monzon O, Rodal AA, Strokopytov B, Almo SC, Goode
BL. (2005) Structural and functional dissection of the Abp1
ADFH actin-binding domain reveals versatile in vivo adapter
functions. Mol Biol Cell. 2005 Jul;16(7):3128-39.
[abstract]
Rodal AA, Kozubowski L, Goode BL, Drubin DG, Hartwig JH.
(2005) Actin and septin ultrastructures at the budding yeast
cell cortex. Mol Biol Cell. 2005 Jan;16(1):372-84.
[abstract]
Rodal, A.A., O. Solkolova, D. Robins, S. Hippenmyer, H.
Riezman, N, Grigorieff, and B.L. Goode. (2005) Conformational
changes in the Arp2/3 complex leading to actin nucleation.
Nat Struct Mol Biol. 2005 Jan;12(1):26-31. Epub 2004
Dec 12. [abstract]
Mattila PK, Quintero-Monzon O, Kugler J, Moseley JB, Almo
SC, Lappalainen P, Goode BL. (2004) A high-affinity interaction
with ADP-actin monomers underlies the mechanism and in vivo
function of Srv2/cyclase-associated protein. Mol Biol
Cell. 2004 Nov;15(11):5158-71. [abstract]
Xu, Y., J.B. Moseley, I. Sagot, F. Poy, D. Pellman, B.L.
Goode, and M.J. Eck (2004). Crystal structures of a formin
homology-2 domain reveal a flexibly tethered Architecture.
Cell 116:1-20. [abstract]
Moseley JB, Sagot I, Manning AL, Xu Y, Eck MJ, Pellman
D, Goode BL. (2004) A conserved mechanism for Bni1- and
mDia1-induced actin assembly and dual regulation of Bni1
by Bud6 and profilin. Mol Biol Cell. 15(2):896-907. [abstract]
Balcer HI, Goodman AL, Rodal AA, Smith E, Kugler J, Heuser
JE, Goode BL. (2003) Coordinated regulation of actin filament
turnover by a high-molecular-weight Srv2/CAP complex, cofilin,
profilin, and Aip1. Curr Biol. 13(24):2159-69. [abstract]
Goodman, A., B.L. Goode, P. Matsuidara, and G. Fink (2003).
The S. cerevisiae calponin/transgelin homologue Scp1p functions
with fimbrin to regulate the actin cytoskeleton. Mol. Biol.
Cell 14:2617-2629. [abstract]
Rodal, A.A., A.L. Manning, B.L. Goode, and D.G. Drubin
(2003). Negative regulation of yeast WASp by two SH3 domain-containing
proteins. Curr. Biol. 13:1-20. [abstract]
Humphries, C., H.I. Balcer, J.L. D'Agostino, B. Winsor,
D.G. Drubin, G. Barnes, B. Andrews, and B.L. Goode (2002).
Direct regulation of Arp2/3 complex activity and function
by the actin binding protein coronin. J. Cell Biol. 159(6):993-1004.
[abstract]
Sagot, I., A.R. Rodal, B.L. Goode, and D. Pellman (2002).
An actin nucleation mechanism mediated by the formin Bni1
and profilin. Nature Cell Biol. 8:626-631. [abstract]
Rodal, A.A., and Goode, B.L. (2001) Modular complexes that
regulate actin assembly in budding yeast. Curr. Opin. Microbiol.
4:703-12. [abstract]
Goode, B. L. Purification of yeast actin and actin-associated
proteins (2002). Guide to yeast genetics and molecular biology.
Methods Enzymol. 351:433-41. (editors: C. Guthrie and G.
Fink).
Goode, B.L., A.A. Rodal, G. Barnes, and D.G. Drubin (2001).
Activation of the Arp2/3 complex by the actin filament binding
protein Abp1p. J. Cell Biol.153(3): 627-634. [abstract]
View Complete Publication List on PubMed:
Bruce Goode
