Aravintham Samuel (Physics)
Howard A. Stone (DEAS)
David A. Weitz (DEAS, Physics)
George M. Whitesides (Chemistry)
Xiaowei Zhuang (Chemistry, Physics)

Collaborators: Howard Berg (Biology, Physics) and N. Michelle Holbrook (Biology)
Number of graduate students: 4
Number of postdoctoral associates: 2

This IRG focuses on the materials science for the study of biological systems at the scale of the cell. It has made considerable progress combining studies of imaging to study the structure and properties of cells and confining cells on controlled structures to investigate the response of the cell. In addition, new methodology was developed to locally disrupt structures within the cell using femtosecond laser light, and this was applied to the investigation of the behavior of neurons in an organism.

Ingber has continued his studies focusing on development and applications of new micromaterials and nanotechniques for analysis of the living mammalian cells. Working in collaboration with Whitesides, he cultured living cells on micropatterned substrates, self-assembled monolayers, and flexible polydimethylsiloxane polymers and used these methods to control the shape and position of cells, as well as regulate their growth, differentiation, contractility, motility, and death. They also developed and applied microfluidic techniques to place cells and molecules in defined patterns and positions and to deliver soluble molecules to precise locations and subcompartments within adherent cells. With Mazur, he used femtosecond lasers to create a nanosurgical technique that allows them to precisely disrupt molecular structures inside living cells without altering surrounding structures or compromising cell viability. With Weitz, he analyzed the biophysical basis of molecular and organelle transport within the cytoplasm. With Prentiss, he applied micromagnetic methods to guide cellular self-assembly into specific tissue patterns, and used micro- and nano-magnetic approaches to probe cellular mechanics and develop magnetically actuatable living cellular “switches.” Through this combination of approaches from microelectronics, micromagnetics, optics, biophysics and molecular cell biology, they are gaining greater insight into the structural basis of cell regulation. These studies will help establish a foundation of knowledge relating to cellular microsystems that will form the basis of tomorrow’s microsensors, biochips, and cell-based microdevices.

The initial step in all cell motility is accomplished by the extension of actin-rich processes called lamellipodia. Ingber has shown that when single cells are cultured on individual cell-sized square adhesive islands created with microcontact printing and coated with fibronectin, these square cells preferentially extend lamellipodia from the corners relative to the sides when stimulated by motility factors. He now has used different shaped islands created with the same micropatterning technique to demonstrate that cells preferentially extend these motile processes from acute, rather than obtuse, angles (Fig. 1), and that they deposit new fibronectin fibrils in their corner regions. The physical determinants that he uncovered are critical for directional migration and may also be used as design criteria for creating engineered substrates that promote tissue regeneration.

Mazur has developed a technique to disrupt submicrometer-sized organelles within living cells or tissue without affecting the surrounding material or compromising viability of the cell or organism. This method, called nanosurgery, received substantial media attention, and was featured in Nature Science Update and The Boston Globe (front page, Nov. 22, 2003). Working with a new member of the MRSEC, Asst. Prof. Samuel, he is studying a nematode worm Caenorhabditis elegans, whose behavior is encoded in the structure and function of its neural network, which comprises exactly 302 neurons that interconnect in the same way in every adult animal. Using tightly focused femtosecond infrared laser pulses, they precisely and reproducibly snip individual wires of an otherwise intact and invariant wiring diagram. By analyzing the behavior of the operated animals, they determine the computational effects of blocking information transmission at specific points.

In a preliminary experiment, they severed fibers of the ASH neuron which plays a crucial role in the osmotic avoidance behavior, nose touch avoidance, and chemotaxis to certain compounds. Severing the ASH neuron should block transmission of sensory information from the sensory cilia to the synaptic connections of the ASH neuron and hence all downstream neurons. Hence, by snipping two wires, belonging to the left and right ASH neurons, they eliminate the behavior underpinned by ASH. They have successfully snipped specific axons without affecting the remainder of the worm (see Fig. 2), and are currently evaluating the effect on behavior. Using laser nanosurgery, they will be able to assign functions to parts of neurons in the worm.

Zhuang has begun to unravel the molecular mechanisms underlying the nuclear trafficking of viral genes. Many viruses deliver their genomes to the cell nucleus for replication and expression. However, little is known about how synthetic gene-delivery materials help targeting foreign genes into the cell. Therefore, efforts to combat viral diseases and to improve gene therapy could both benefit from new experimental techniques for investigating the nuclear trafficking of genetic materials. She has developed a physical technique to study gene trafficking by tracking single genetic carriers in living cells using sensitive fluorescence microscopy, and has applied this technique to study the nuclear trafficking of influenza genes, packaged in the form of viral ribonucleoproteins (vRNPs) (Fig. 3). Single-vRNP trajectories show that vRNPs are transported in the cell by diffusion. She has identified two distinct types of interactions between the vRNPs and the nuclear envelope. Her experiments provide new insights into the regulation mechanisms for the nuclear import of vRNPs; the influenza M1 protein down-regulates the nuclear import of vRNP by inhibiting the binding of vRNPs to the nuclear envelope. This single-gene tracking approach will find broad application in the investigation of genetic trafficking in cells.

Prentiss demonstrated that the unzipping of heterogeneous double stranded DNA proceeds by a series of jumps, as predicted earlier by Nelson. This experiment demonstrated that thermal fluctuations result in variations in the number of unzipped base pairs as a function of time, even for identical molecules under identical conditions. She also made the first measurement of the phase diagram for unzipping of dsDNA in the force temperature plane. Nelson’s theory correctly predicts the critical force at physiological temperatures, but departs strongly from the observations at temperatures outside of the physiological range. They believe that hairpin formation in the ssDNA and bubble formation in the dsDNA reduce the free energy difference between ssDNA and dsDNA at high temperatures, allowing the strands to unzip at lower forces than predicted.

Weitz is studying the physical properties of networks of entangled and crosslinked actin, a semi-flexible polymer that is a major constituent of the cellular cytoskeleton. The mechanical properties of these semi-flexible polymer networks are determined by a competition of entropic and enthalpic effects and serve as an intermediate between classical networks of flexible polymers and rigid rods. Embedding colloidal particles in the F-actin networks and measuring their thermal motion probes the microstructure and mechanics of network. In collaboration with Reichman and an REU student, they probed spatial and temporal fluctuations in the network microstructure and proposed a framework in which to understand these in terms of the thermally driven fluctuations of single actin filaments. To further develop the applicability of microrheology, he collaborated with Whitesides to develop new coatings for the tracer particles used to probe biological samples. They developed a robust protocol for binding short poly(ethylene glycol) (PEG) chains to the surfaces of colloidal particles. These PEG-coated beads resist protein adsorption onto the colloid surface and enable precise characterization of bio-materials using microrheology techniques.