Actin filament lengths are precisely regulated and very stable in the red blood cell (RBC) membrane skeleton, while in platelets, actin filament lengths are dynamically regulated during receptor- mediated actin assembly as platelets extend lamellipodia during thrombogenesis. The broad, long term objective of this research is to elucidate how actin filament length and turnover is controlled in RBCs and platelets. An additional objective is to determine the role of actin filament length regulation in RBC membrane skeleton biogenesis and stability, and how aberrant filament length regulation may result in abnormal RBCs and hemolytic anemias. This proposal focuses on molecular mechanisms and in vivo functions of tropomodulins (Tmods), tropomyosin (TM)-regulated actin filament pointed end-capping proteins in RBCs (Tmod1) and platelets (Tmod3).
The specific aims are: (1) To investigate the molecular basis for Tmod1 or Tmod3 binding to RBC or platelet TMs, and for TM-regulated actin capping. Biochemical and biophysical assays will be used to identify TM isoform-specific binding and TM-actin capping domains and a novel Tmod3 monomer-binding site. (2) To establish the in vivo function of Tmod1 in regulation of RBC actin filament length, membrane skeleton assembly and stability using mouse models. Tmod1 and Tmod3 conditional knockout mice will be generated followed by breeding with a ?LCR-?pr- Cre mouse to produce single or double knockouts for Tmod1 and/or Tmod3 in RBCs and platelets. RBC phenotypes will be examined by hematology, ektacytometry, membrane skeleton assembly, and electron microscopy of actin filament lengths in membrane skeletons. (3) To investigate an in vivo function for Tmod3 in receptor-mediated actin assembly during activation and spreading of platelets. Platelet function in Tmod3-deficient mouse platelets will be evaluated by hematological analyses, bleeding times, and thrombus formation under flow ex vivo. A role for Tmod3 in actin assembly, shape change and spreading will be evaluated by biochemical assays, immunofluorescence and electron microscopy, and by time-lapse interference reflection and fluorescence microscopy of lamellipodia and filopodia protrusion in living platelets. Molecular requirements for Tmod3 function will be tested using a permeabilized platelet model for receptor-coupled platelet activation, by introduction of Tmod3 or Tmod1 domains and mutants. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
9R01HL083464-20
Application #
7034274
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Sarkar, Rita
Project Start
1984-12-01
Project End
2010-11-30
Budget Start
2006-01-20
Budget End
2006-11-30
Support Year
20
Fiscal Year
2006
Total Cost
$597,832
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Smith, Alyson S; Nowak, Roberta B; Fowler, Velia M (2018) High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure. Methods Mol Biol 1698:205-228
Gokhin, David S; Fowler, Velia M (2017) Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images. J Microsc 265:11-20
Sui, Zhenhua; Gokhin, David S; Nowak, Roberta B et al. (2017) Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells. Mol Biol Cell 28:2531-2542
Nowak, Roberta B; Papoin, Julien; Gokhin, David S et al. (2017) Tropomodulin 1 controls erythroblast enucleation via regulation of F-actin in the enucleosome. Blood 130:1144-1155
Wu, Tongbin; Mu, Yongxin; Bogomolovas, Julius et al. (2017) HSPB7 is indispensable for heart development by modulating actin filament assembly. Proc Natl Acad Sci U S A 114:11956-11961
Fowler, Velia M; Dominguez, Roberto (2017) Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles. Biophys J 112:1742-1760
Gokhin, David S; Fowler, Velia M (2016) Feisty filaments: actin dynamics in the red blood cell membrane skeleton. Curr Opin Hematol 23:206-14
Gokhin, David S; Ochala, Julien; Domenighetti, Andrea A et al. (2015) Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle. Development 142:4351-62
Fischer, Robert S; Fowler, Velia M (2015) Thematic Minireview Series: The State of the Cytoskeleton in 2015. J Biol Chem 290:17133-6

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