Macromolecular biophysics of the cytoskeleton

Endothelial cells engineered to express GFP-tubulin (green) and subsequently fixed and stained for F-actin (red) and paxillin (blue). Note that cell shape is largely defined by the structure of the cytoskeleton, and that the morphology of each filament system is dramatically different, reflecting the fact that each filament system bears and exerts different types of mechanical loads.

The living cell is a complex entity whose remarkable, emergent capacity to sense, integrate, and respond to environmental cues relies on an intricate series of interactions among the cell's macromolecular components. The establishment and maintenance of cell shape illustrates this point well. Contrary to the classical picture painted by many textbooks, cells are not membrane-bound aqueous vessels in which biochemical reactions occur in the dilute limit. Instead, the cytoplasm of a living cell is dominated by a viscoelastic network of structural elements known collectively as the cytoskeleton. The cytoskeletal lattice, which consists of microfilaments (actin filaments and contractile actomyosin filaments), microtubules, and intermediate filaments, is directly responsible for determining cell shape, generating mechanical forces, resisting externally imposed forces, and transducing extracellular biochemical and mechanical stimuli to the cytoplasm. Cytoskeletal dynamics enable the remodeling that is necessary for cell migration and chemotaxis that underly tissue development, the inflammatory response, and tumor invasion and metastasis.

Energy dependence of material removal by a laser nanoscissor. This endothelial cell nucleus has been incised in a series of vertical lines of increasing pulse energy and then visualized by transmission electron microscopy. As the energy rises, more nuclear material is removed (from Heisterkamp et al., 2005).

Cell shape also can determine cell fate independently of the formation of adhesive contacts with surrounding cells or the extracellular matrix (ECM). By using the cytoskeleton as a vehicle for signal transduction, mechanical forces play a central role in driving a wide variety of physiological events including sound processing by hair cells, mechanosensation in the skin, and remodeling of myocardial tissue. Human disease may arise from excessive input to the mechanotransducing cell (e.g., hypertensive hypertrophic cardiomyopathy), genetic lesions in the mechanotransduction apparatus (e.g., hearing loss associated with muscular dystrophy), or alteration in cell or tissue mechanics. Indeed, pharmaceuticals which target cytoskeletal elements are in active use or clinical trials in a variety of disease settings including cancer chemotherapy, treatment of hypertension and inhibition of angiogenesis; mechanical manipulation is also the basis of several therapies (e.g., orthopedic traction, tissue expanders, acupuncture).

Energy dependence of material removal by a laser nanoscissor. This endothelial cell nucleus has been incised in a series of vertical lines of increasing pulse energy and then visualized by transmission electron microscopy. As the energy rises, more nuclear material is removed (from Heisterkamp et al., 2005).

We study how the physical properties of individual cytoskeletal polymers enable these materials to form a three-dimensional, mechanoresistive network in cells. In pursuing this goal, we biophysically characterize purified cytoskeletal proteins with a variety of tools, observe and probe the dynamics of individual cytoskeletal polymers in living cells, and complement our studies with computational analysis and simulation. For example, our previous work with neurofilaments (NFs) included atomic force microscopy of purified NFs, statistical analysis of intracellular NF distributions, and polymer-based Monte Carlo simulations. This work has helped create a model for NF organization in which adjacent NFs interact through phosphate-dependent steric interactions mediated by their unstructured sidearm domains. We are continuing this work and applying the methods to other macromolecular systems.

Incision of a microtubule in a living cell with a femtosecond laser nanoscissor. When the microtubule is severed by applying a short series of focused, femtosecond laser pulses, it straightens due to the release of elastic energy and then rapidly depolymerizes (from Heisterkamp et al., 2005).