Abstract

This is an interdisciplinary Project between applied physicists, and structural and cardiovascular biologists. This collaboration will develop and implement the methodology required for imaging the elemental composition of cell membranes and organelles at 2nm resolution and protein structure at 1nm resolution. Major objectives to be achieved through these methods include: mapping functionally distinct intracellular Ca2+-storage sites in vascular smooth muscle and endothelial cells; quantitatively localizing the concentration of Ca-bound to cardiac intercalated discs, gap junctions and to non-specialized regions of the plasma membrane and determining the chemical (sulfur containing basement membrane and determining the chemical (sulfur containing basement membrane or phosphorus containing phospholipid) components associated with bound Ca. A 200kV field emission gun (FEG) equipped electron microscope, available for the first time, will be used for X-ray mapping and scanning transmission energy filtered electron microscopy (STEFEM) required for compositional imaging of biological cryosections. Quantitative methods of signal collection and data analysis will be developed for biological electron energy loss spectroscopy (EELS). Tissues will be prepared by rapid freezing and cryoultramicrotomy. The quantitative localization of Ca2+-bound to cardiac membranes is expected to provide information about the Ca2+-dependence of normal and abnormal cardiac conduction. The compositional imaging methods to be developed will provide a unique and general approach to a broad range of physiological and pathological biomedical problems, and represent a major significant advance in analytical electron microscopy. The 200kV microscope will also be used for spot scan imaging of Ca2+-ATPase (Project 1), the problems of intracellular Ca2+-storage and Ca2+-binding sites on cardiac membranes are intimately related to the aims of Project 3, and this Project (5) will also provide electron microscopic support to Project 2.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL048807-03
Application #
3759108
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
University of Virginia
Department
Type
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904

  Publications

Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya et al. (2012) Insights into the inhibition of the p90 ribosomal S6 kinase (RSK) by the flavonol glycoside SL0101 from the 1.5 Å crystal structure of the N-terminal domain of RSK2 with bound inhibitor. Biochemistry 51:6499-510
Hoofnagle, Mark H; Neppl, Ronald L; Berzin, Erica L et al. (2011) Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes. Am J Physiol Heart Circ Physiol 300:H1707-21
Jin, Li; Gan, Qiong; Zieba, Bartosz J et al. (2010) The actin associated protein palladin is important for the early smooth muscle cell differentiation. PLoS One 5:e12823
Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S et al. (2009) Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction. J Biol Chem 284:21569-79
Jin, Li; Hastings, Nicole E; Blackman, Brett R et al. (2009) Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury. J Muscle Res Cell Motil 30:41-55
Cierpicki, Tomasz; Bielnicki, Jakub; Zheng, Meiying et al. (2009) The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA. Protein Sci 18:2067-79
Jin, Li; Yoshida, Tadashi; Ho, Ruoya et al. (2009) The actin-associated protein Palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies. J Biol Chem 284:2121-30
Zheng, Meiying; Cierpicki, Tomasz; Momotani, Ko et al. (2009) On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF. BMC Struct Biol 9:36
Jelen, Filip; Lachowicz, Pawel; Apostoluk, Wlodzimierz et al. (2009) Dissecting the thermodynamics of GAP-RhoA interactions. J Struct Biol 165:10-8
Freitas, Maria Regina; Eto, Masumi; Kirkbride, Jason A et al. (2009) Y27632, a Rho-activated kinase inhibitor, normalizes dysregulation in alpha1-adrenergic receptor-induced contraction of Lyon hypertensive rat artery smooth muscle. Fundam Clin Pharmacol 23:169-78

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