Homepage of Liang Cai

Publication

» Liang CAI, Alexander M Makhov, Dorothy A Schafer and James E Bear. Coronin 1B antagonizes Cortactin and remodels Arp2/3-containing actin branches in lamellipodia. Cell [J], 2008, 134(5):828-842.
[The-Cover, Listed, Recommended in F1000, Featured by Cell Migration Gateway; PMID 18775315]

» Liang CAI and James E Bear. Peering deeply inside the branch. Journal of Cell Biology [J], 2008, 180(5):853-855.
[comment on Rouiller et al. 2008; PMID 18316414]

» Liang CAI, Alexander M Makhov, and James E Bear. F-actin binding is essential for coronin 1B function in vivo. Journal of Cell Science [J], 2007, 120(10):1779-1790.
[The-Cover, Listed; PMID 17456547]

» Liang CAI, Thomas W Marshall, Andrea C Uetrecht, Dorothy A Schafer, James E Bear. Coronin 1B coordinates Arp2/3 complex and Cofilin activities at the leading edge. Cell [J], 2007, 128(5):915-929.
[Listed, Highlighted in Nature, Recommended in F1000; PMID 17350576]

» Thomas W Marshall, Liang CAI, James E Bear. Chemistry comes to the cell: ASCB 2005. Nature Chemical Biology [J], 2006, 2(3):119-122.
[PMID 16484998]

» Liang CAI, Nicholas Holoweckyj, Michael D Schaller, James E Bear. Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility. The Journal of Biological Chemistry [J], 2005, 280(36): 31913-31923.
[PMID 16027158]

Meeting Abstracts

» Coronin 1B Debranches Actin Filaments Generated by the Arp2/3 Complex
L. Cai,^1,2 A. M. Makhov,³ D. A. Schafer,⁴ J. E. Bear^1,2;
¹Department of Cell and Developmental Biology, UNC-Chapel Hill, Chapel Hill, NC, ²Lineberger Comprehensive Cancer Center, Chapel Hill, NC, ³Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, ⁴Department of Biology and Cell Biology, University of Virginia, Charlottesville, VA
Presentation Number: 1014-ASCB2007 (not show up due to conflict with my thesis defense schedule)
Actin cytoskeleton assembly and remodeling are complex processes that require precise spatial and temporal control of actin and numerous actin-binding proteins. Coronins are highly conserved actin binding proteins that have been implicated in numerous cellular processes such as chemotaxis and fibroblast motility. In lamellipodia, current experimental evidence suggests that Coronin 1B coordinates Arp2/3 complex and Cofilin activities and plays a critical role in regulating actin dynamics. Despite the strong phenotypes observed upon Coronin 1B depletion in vivo, recombinant Coronin 1B exhibits modest inhibition of Arp2/3 nucleation in pyrene actin assays. To elucidate the molecular mechanism of Coronin 1B’s inhibition on Arp2/3 complex activity, we utilized TIRF microscopy to study Arp2/3-mediated actin branch formation in real time. Using this technique, we observed much slower total actin filament growth and much higher frequency of de-branching events in the presence of Coronin 1B than in controls. We developed an in vitro biochemical assay to analyze Coronin 1B’s effect on de-branching. Wild-type Coronin 1B not only de-branches Arp2/3 actin filaments, but also disrupts the interaction between the Arp2/3 complex and the sides of actin filaments (IC₅₀, 73 nM). Using specific Coronin 1B mutants, we observed that both F-actin and Arp2/3 binding activity are required for this effect. Interestingly, we found that Cortactin enhances Arp2/3's side-binding (IC₅₀, 51 nM) in this assay. Consistent with previous data, we observed that Cortactin stabilizes the actin branches in TIRF analysis. Finally, we found that Coronin 1B antagonizes Cortactin in pyrene-actin assays. These data demonstrate that Coronin 1B is the first known protein with specific Arp2/3 de-branching activity, and that Coronin 1B is a direct antagonist of Cortactin in actin dynamics.

» Coronin 1B Coordinates Arp2/3 and Cofilin Activity at the Leading Edge
J. E. Bear,¹ L. Cai,¹ T. W. Marshall,¹ A. C. Uetrecht,¹ D. A. Schafer²;
¹Cell & Developmental Biology, UNC-Chapel Hill, Chapel Hill, NC, ²Biology, University of Virginia, Charlottesville, VA
Presentation Number: 722-ASCB2006
Talk: Minisymposium 14 - Regulation of the Cytoskeleton
Arp2/3-dependent actin filament nucleation and Cofilin-driven filament turnover are two major factors that control the dynamics of lamellipodia in motile cells, but the coordination of these activities is poorly understood. Coronins are highly conserved F-actin binding, WD repeat proteins that have been implicated in cell motility in model organisms. Yeast Coronin inhibits Arp2/3 complex in vitro, but the function of Coronins in mammalian cells is not known. We report that human Coronin 1B inhibits actin nucleation by the Arp2/3 complex and that this inhibition is regulated by the Slingshot-1L (SSH1L) phosphatase via the dephosphorylation of Serine 2 on Coronin 1B. Furthermore, we find that SSH1L, Coronin 1B and Arp2/3 exist in a ternary complex in vivo that is bridged by Coronin 1B. Functional studies of lamellipodial dynamics indicate that depletion of Coronin 1B alters lamellipodial protrusion and that Coronin 1B is required for enhanced ruffling at the cell periphery induced by expression of SSH1L. In addition to being a substrate for SSH1L, Coronin 1B is required for proper targeting of SSH1L to lamellipodia where it likely regulates filament turnover by activating Cofilin. Consistent with this idea, depletion of Coronin 1B increased cellular levels of phospho-Cofilin and expression of an activated Cofilin mutant partially suppressed the effects of Coronin 1B knockdown on lamellipodia dynamics. Together, our data suggest that Coronin 1B coordinates Arp2/3 and Cofilin activity at the leading edge.

» Serine 2 Phosphorylation Regulates Coronin 1B Activity and Affects Cell Migration
L. Cai,^1,2 M. D. Schaller,^1,2 J. E. Bear^1,2;
¹Cell and Developmental Biology, School of Medicine, UNC at Chapel Hill, Chapel Hill, NC, ²Lineberger Comprehensive Cancer Center, Chapel Hill, NC
Presentation Number: 2444-ASCB2005
Poster Board Number: B215-ASCB2005
Coronin 1B, a conserved WD40 repeat-containing, actin binding protein, is ubiquitously expressed and co-localizes with the Arp2/3 complex at the leading edge of fibroblasts. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated through protein kinase C (PKC) phosphorylation. Using tryptic peptide mapping and site-direct mutagenesis, we show that Coronin 1B is phosphorylated at Serine 2 (Ser2) upon phorbol-12-myristate-13-acetate (PMA) stimulation in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to PMA and increased speed in single cell tracking assay, while the S2D mutant expression attenuates PMA-induced ruffling and slows cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts through Ser2 phosphorylation, and this phosphorylation is responsible for a measurable fraction of PMA's effects on motility. To elucidate the regulation of Coronin 1B function via Ser2 phosphorylation, we developed an assay for measuring dephosphorylation using a phosphor-specific antibody. Following termination of PMA treatment with PKC inhibitors, phosphorylated Coronin 1B undergoes a rapid, okadaic acid-insensitive dephosphorylation and returns to basal phosphorylation level within 10 min. RNAi-mediated knockdown of Coronin 1B leads to defects in both leading edge protrusion and cell motility, which can be rescued by over-expression extrinsic wild-type protein. While S2A and S2D mutants reciprocally enhance or suppress cell motility in the presence of wild-type protein, neither is capable of rescuing Coronin 1B knockdown phenotypes. These data are consistent with a model in which Coronin 1B undergoes cycles of phosphorylation and dephosphorylation in vivo in order to function properly.

» Coronin 1B interacts with Arp2/3 and is regulated by PKC
L. Cai, N. Holoweckyj, M. D. Schaller, J. E. Bear;
Cell and Developmental Biology, Lineberger Cancer Center, School of Medicine, UNC at Chapel Hill, NC
Presentation Number: 1495-ASCB2004
Poster Board Number: B141-ASCB2004
Cancer metastasis, embryonic development, and many other processes rely on actin-based cell migration. Coronins are a family of WD40 repeat-containing actin binding proteins first discovered in Dictyostelium. Coronin 1B is a ubiquitously expressed member of the mammalian Coronin family that is localized to the leading edge, actin stress fibers and endocytic structures in fibroblasts. Molecular modeling indicates that the WD repeats of Coronin 1B adopt a beta barrel structure, but that conserved regions on either side of the five canonical WD repeats each contribute an additional blade to the structure. This model strongly suggests that Coronins have a seven bladed beta barrel structure, rather than the five-bladed model postulated by earlier studies. Coronin 1B co-localizes with the Arp2/3 complex at the leading edge suggesting that these proteins may co-operatively regulate cytoskeleton organization. Co-immunoprecipitation confirms the interaction between Coronin 1B and Arp2/3. Previous work suggested that Coronins may be regulated by PKC phosphorylation. Recombinant Coronin 1B is robustly phosphorylated by PKC alpha in vitro. Metabolic labeling of HEK293 cells with radioactive orthophosphate shows that Coronin 1B is phosphorylated in response to PMA stimulation in vivo. Using tryptic peptide mapping and mutagenesis, we have identified a single site on Coronin 1B that is phosphorylated by PKC in vivo. Initial data indicate that PKC phosphorylation of Coronin 1B regulates its ability to partition between a cytosolic pool and an actin-associated pool.