Cell and Developmental Biology University of Michigan

Research

Emergent behavior of multiple molecular motors; Protein engineering of FRET bio-sensors; Signaling pathways in cardiac hypertrophy

Research Topics

The laboratory research spans the fields of single molecule biophysics, protein biochemistry and cell biology and integrates a variety of experimental and computational approaches.

Figure 1
Coordinated movement of myosin VI-coated nanospheres on the dense F-actin network in the fish epidermal keratocyte lamellipodium. The linear trajectories result from flexible elements in the myosin protein that enable multiple dimers or monomers of this motor to coordinate their interactions and move cargo over large distances (> 10 μ m).

Coordinated function of the myosin family of molecular motors

Myosins are actin-based molecular motors implicated in diverse cellular processes. They are cellular engines that convert the chemical energy derived from the hydrolysis of ATP to mechanical motion. A lot is known about the structure and function of myosin molecules and the interaction of single myosins with single actin filaments. Myosin function in vivo is an emergent property of the simultaneous interaction of multiple myosins with multiple actin filaments. These coordinated interactions are essential for cellular processes such as membrane trafficking, mRNA transport and maintenance of membrane tension. An unexplored frontier in molecular motor research is how the same motor interacts with different subcellular compartments to perform different cellular functions. The laboratory uses a model experimental system (Figure 1), developed by Dr. Sivaramakrishnan, to examine the movement of organelle-sized nanostructures linked to different numbers and types of myosin motors on an organized meshwork of actin filaments.

Figure 2
Schematic of a SPASM based FRET sensor (a) Inactive (Open - No FRET (Fluorescence resonance energy transfer)) and (b) Active (Closed - FRET) state. Illustration here shows protein calmodulin (CAM) and its binding peptide, separated by an ER/K α-helix. Interaction between CAM and peptide in its closed state results in FRET between CFP and YFP. ER/K α-helix stiffness is engineered, such that FRET efficiency changes with intracellular protein concentration.

Regulation of protein-protein interaction using an ER/K α-helix

Cell biological processes in health and disease are regulated by dynamic interactions between proteins. We use a technique termed SPASM (Systematic Protein Affinity Strength Modulation) to regulate the strength of protein-protein interactions in vivo. SPASM involves two interacting proteins separated by an ER/K helix of designed mechanical stiffness. The ER/K helix acts as a semi-flexible structure that regulates the strength of interaction between the proteins it separates. The mechanical properties of the helix can be engineered systematically to alter the affinity of the protein-protein interaction. The laboratory is focused on the use of SPASM for a variety of applications including the design of FRET bio-sensors, protein concentration sensors (Figure 2) and modulating autoinhibition of enzymes such as kinases.

Figure 3
Schematic of single-cell stretch device to study force-length changes in single cardiomyocytes.

Contractility and signaling in cardiomyocytes

Cardiomyocytes have a highly organized acto-myosin cytoskeleton that drives contractility of heart muscle. Contractility of cardiomyocytes is influenced by numerous factors including the control of sarcomeric proteins by calcium signaling. Genetic mutations in sarcomeric proteins such as β-cardiac myosin can also influence contractility in diseases such as β-cardiac myosin. Recent technological advances have enabled the contractility measurements of isolated cardiomyocytes under physiological load conditions (Figure 3). The laboratory is broadly interested in the development of FRET bio-sensors to act as spatio-temporal probes of signaling events in cardiomyocytes during normal and disease states.

Lab Members

• Rizal Fajar Hariadi
  Research Fellow
• Ritt Mike
  Research Lab Technician
• Rabia Malik
  Graduate Student
• Mario Cale
  Undergradate Student
• Kendal Noonan
  Undergradate Student
• Terrence Tigney
  Undergradate Student
• William Wang
  Undergradate Student

Publications

Representative Publications

1. Set up of a dual-beam optical trap to study the myosin family of molecular motors , J. Sung, S. Sivaramakrishnan, A. Dunn, J.A. Spudich, Methods in Enzymology, vol 475, 321-75, 2010
2. Revelations from myosin VI: the most innovative of molecular motors , S. Sivaramakrishnan, J.A. Spudich, Nature Reviews: Molecular and Cellular Biology, vol 11(2), 128-37, Feb 2010
3. Combining single molecule optical trapping and small angle X-ray scattering measurements to compute the persistence length of a protein ER/K a-helix , S. Sivaramakrishnan, J. Sung, M. Ali, S. Doniach, H. Flyvbjerg, J. A. Spudich, Biophysical Journal, vol 97, 1-7, Dec 2009, 2993-9
4. Coupled myosin VI motors facilitate unidirectional movement on an F-actin network , S. Sivaramakrishnan, J.A. Spudich, Journal of Cell Biology, vol. 187, no. 1, 53-60, Oct 5 2009
5. Insights into human b-cardiac myosin function from single molecule and single cell studies , S. Sivaramakrishnan, E. Ashley, L. Leinwand, J.A. Spudich, Journal of Cardiovascular Translational Research (Springer New York), Invited Review, Epub Sep. 25th, 2009
6. Dynamic charge interactions create surprising rigidity in the ER/K a-helical protein motif , S. Sivaramakrishnan, B. J. Spink, A. Y. L. Sim, S. Doniach, J. A. Spudich, Proceedings of the National Academy of Sciences, USA, vol. 105, no. 36, 13356-61, Sep 9 2008
7. Bridging the Gap between the Structure and Function of Myosin VI , B.J. Spink, S. Sivaramakrishnan, J. Lipfert, S. Doniach, J.A. Spudich, Nature: Structural and Molecular Biology, vol. 15, no. 6, 591-97, Jun 2008
8. Shear stress induced reorganization of the keratin intermediate filament network requires PKC ζ , S. Sivaramakrishnan, J.L. Schneider, A. Sitikov, R.D. Goldman, K.M. Ridge, Molecular Biology of the Cell, 2009, April 8^th Epub, vol 20, no 11, 2755-65
9. Micromechanical properties of keratin intermediate filament networks , S. Sivaramakrishnan, J.V. DeGuilio, R.D. Goldman, J.N. Schneider, K.M. Ridge, Proceedings of the National Academy of Sciences, USA, vol. 105, no. 3, 889-894, Jan 22 2008