The long term objectives of the proposed research are to understand how the proteins responsible for motility in cells are organized into contractile units and what factors control the formation and function of these units. The specific goals seek to use fluorescently labeled contractile proteins to investigate how stress fibers, cleavage rings and myofibrils are formed in living cells; to examine the interactions of four different actin-binding proteins with stress fibers and myofibrils in living and permeabilized cells; and to extend current knowledge of stress fiber and cleavage ring ultrastructure. The major focus of our proposal is on microinjection studies using fluorescently labeled proteins. Myosin, actin, alpha-actinin and tropomyosin will be prepared from both muscle and non-muscle sources, labeled with different fluorescent dyes and microinjected into a variety of cultured epithelial and fibroblastic cells as well as embryonic myocytes in culture and in situ. Actin will be injected as profilactin and in the uncomplexed monomer form, G-actin. The length of time required for incorporation and the longevity of the incorporated protein will be compared for the same protein prepared from different sources and using different fluorescent dyes. The ability of the labeled proteins to cycle as stress fibers, cleavage rings and myofibrils assemble and disassemble will be examined using low-light level cameras coupled with video-enhanced microscopy. Photo-bleaching experiments using an argon laser will be used to measure the recovery times of labeled proteins into stress fibers, cleavage furrows and myofibrils and to determine whether or not addition occurs as a treadmilling process. Two actin-binding proteins, vitamin D binding protein and brain capping protein, will be reacted with isolated myofibrils and permeabilized non-muscle cells to determine whether they interfere with the binding of exogenous contractile proteins to these systems. In addition, spectrin and filamin will be purified and labeled with fluorescent tags to determine if they bind to the permeabilized systems. Subsequently, these four proteins will be injected into living cells so that their interactions with stress fibers, cleavage furrows and myofibrils can be examined. The ultrastructure of stress fibers and cleavage rings will be studied using gold labeled antibodies against contractile proteins and actin-binding proteins such as filamin. The information gained in these studies will provide basic knowledge of motility in normal cells and provide a framework against which to compare motility in cells involved in processes such as wound healing or cancer in which transformed cells lack stress fibers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025653-10
Application #
3273183
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-09-15
Project End
1989-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Sanger, J M; Dabiri, G; Mittal, B et al. (1990) Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I. Proc Natl Acad Sci U S A 87:5474-8
Zurek, B; Sanger, J M; Sanger, J W et al. (1990) Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells. J Cell Sci 97 ( Pt 2):297-306
Haddad, J G; Harper, K D; Guoth, M et al. (1990) Angiopathic consequences of saturating the plasma scavenger system for actin. Proc Natl Acad Sci U S A 87:1381-5
Sanger, J M; Dome, J S; Mittal, B et al. (1989) Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells. Cell Motil Cytoskeleton 13:301-19
Hock, R S; Sanger, J M; Sanger, J W (1989) Talin dynamics in living microinjected nonmuscle cells. Cell Motil Cytoskeleton 14:271-87
Mittal, B; Sanger, J M; Sanger, J W (1989) Visualization of intermediate filaments in living cells using fluorescently labeled desmin. Cell Motil Cytoskeleton 12:127-38
Das, M; Chauhan, S S; Mishra, V S et al. (1989) Aberrant postendocytotic fate of a 34-kDa molecular mass growth factor from human trophoblasts. Cancer Res 49:2761-5
Sanger, J M; Mittal, B; Dome, J S et al. (1989) Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells. Cell Motil Cytoskeleton 14:201-19
Dome, J S; Mittal, B; Pochapin, M B et al. (1988) Incorporation of fluorescently labeled actin and tropomyosin into muscle cells. Cell Differ 23:37-52
Ostrovsky, D; Sanger, J W; Lash, J W (1988) Somitogenesis in the mouse embryo. Cell Differ 23:17-25

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