Rearrangements of the actin cytoskeleton are critical to numerous cellular processes and are defective in many genetic and infectious diseases. Members of the Wiskott-Aldrich Syndrome Protein (WASP) family play key roles in controlling actin dynamics throughout biology. In the previous period we discovered a new mechanism of WASP family regulation that unified a disparate body of unexplained data under a common framework. We also reconstituted two pentameric assemblies, the 400 kDa WRC and the 550 kDa SHRC, that contain and control the WASP proteins WAVE and WASH, respectively. Our WRC crystal structure explained inhibition of WAVE within the assembly. Our biochemical analyses resolved a long-standing dispute regarding WRC activity. Here, we will exploit our unique access to recombinant WRC and SHRC to understand structurally and biochemically how these assemblies respond in complex fashion to upstream stimuli to promote cell migration, neuronal adhesion and vesicle trafficking. We will determine the crystal structure of the WRC bound to the Rac GTPase. The structure, plus complementary biochemical and collaborative cell biological studies, will explain how the WRC is cooperatively activated by GTPases, phospholipids and kinases. We will characterize a novel WRC-binding motif (WIPS) that we have discovered in 30 neuronal adhesion receptors, including many members of the enigmatic protocadherin family. We will determine the structure of a WRC-WIPS complex and learn how various WIPS-containing receptors cooperate with Rac to activate the WRC in vitro and in cells. Finally, we will learn how the SHRC is recruited to membranes through multivalent binding to the retromer coat complex, and the functional consequences of these interactions on actin assembly. Our work will allow the first physical comparisons between WASP proteins that function as single chains (WASP/N-WASP) and those that function within multi-component assemblies (all other family members), revealing new and general principles of signal integration that span the family. We will learn how protocadherins and other neuronal receptors communicate to actin and are coordinated with other signaling inputs as part of their poorly understood adhesive functions. Our findings will provide new reagents and concepts to guide neuroscientists in understanding these receptors in cels and organisms. We wil gain new insights into diseases including autism, epilepsy and deafness, which can be caused by protocadherin mutations. Finally, our studies of the SHRC will address a broadly significant problem in cell biology--how actin assembly and vesicle coat formation are cordinated during endocytic traficking-through a new hypothesis, that multivalency provides a mechanism for SHRC recruitment to respond to retromer density on membranes. This work will suggest general mechanisms by which multivalent interactions can be used to control the specificity and timing of membrane interactions of soluble species.

Public Health Relevance

Our research focuses on understanding control of actin dynamics by members of the Wiskott-Aldrich Syndrome Protein (WASP) family. These molecules are critically involved in many normal biological processes and in numerous diseases, including metastatic cancer, immune disorders, infection, and as suggested by our recent findings, neuronal diseases including autism, epilepsy, deafness and blindness. An understanding of how WASP proteins function will reveal new, general principles in basic biology and could lead to new agents for the diagnosis and treatment of many diseases.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM056322-18
Application #
8643790
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Chen, Baoyu; Brinkmann, Klaus; Chen, Zhucheng et al. (2014) The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell 156:195-207
Banjade, Sudeep; Rosen, Michael K (2014) Phase transitions of multivalent proteins can promote clustering of membrane receptors. Elife 3:
Chen, Baoyu; Padrick, Shae B; Henry, Lisa et al. (2014) Biochemical reconstitution of the WAVE regulatory complex. Methods Enzymol 540:55-72
Chia, Poh Hui; Chen, Baoyu; Li, Pengpeng et al. (2014) Local F-actin network links synapse formation and axon branching. Cell 156:208-20
Chen, Xing Judy; Squarr, Anna Julia; Stephan, Raiko et al. (2014) Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton. Dev Cell 30:569-84
McGough, Ian J; Steinberg, Florian; Jia, Da et al. (2014) Retromer binding to FAM21 and the WASH complex is perturbed by the Parkinson disease-linked VPS35(D620N) mutation. Curr Biol 24:1670-6
Zhao, Huaying; Brautigam, Chad A; Ghirlando, Rodolfo et al. (2013) Overview of current methods in sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Curr Protoc Protein Sci Chapter 20:Unit20.12
Zahm, Jacob A; Padrick, Shae B; Chen, Zhucheng et al. (2013) The bacterial effector VopL organizes actin into filament-like structures. Cell 155:423-34
Hao, Yi-Heng; Doyle, Jennifer M; Ramanathan, Saumya et al. (2013) Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination. Cell 152:1051-64
Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun et al. (2012) Phase transitions in the assembly of multivalent signalling proteins. Nature 483:336-40

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