Fibroblast extracellular matrix and adhesion on microtextured polydimethylsiloxane scaffolds.

The immediate physical and chemical surroundings of cells provide important biochemical cues for their behavior. Designing and tailoring biomaterials for controlled cell signaling and extracellular matrix (ECM) can be difficult due to the complexity of the cell-surface relationship. To address this issue, our research has led to the development of a polydimethylsiloxane (PDMS) scaffold with defined microtopography and chemistry for surface driven ECM assembly. When human fibroblasts were cultured on this microtextured PDMS with 2-6 µm wide vertical features, significant changes in morphology, adhesion, actin cytoskeleton, and fibronectin generation were noted when compared with cells cultured on unmodified PDMS. Investigation of cellular response and behavior was performed with atomic force microscopy in conjunction with fluorescent labeling of focal adhesion cites and fibronectin in the ECM. Changes in the surface topography induced lower adhesion, an altered actin cytoskeleton, and compacted units of fibronectin similar to that observed in vivo. Overall, these findings provide critical information of cell-surface interactions with a microtextured, polymer substrate that can be used in the field of tissue engineering for controlling cellular ECM interactions.

How changes in extracellular matrix mechanics and gene expression variability might combine to drive cancer progression.

Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation.

Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts.

It was our objective to study the role of mechanical stimulation on fibronectin (FN) reorganization and recruitment by exposing fibroblasts to shear fluid flow and equibiaxial stretch. Mechanical stimulation was also combined with a Rho inhibitor to probe their coupled effects on FN. Mechanically stimulated cells revealed a localization of FN around the cell periphery as well as an increase in FN fibril formation. Mechanical stimulation coupled with chemical stimulation also revealed an increase in FN fibrils around the cell periphery. Complimentary to this, fibroblasts exposed to fluid shear stress structurally rearranged pre-coated surface FN, but unstimulated and stretched cells did not. These results show that mechanical stimulation directly affected FN reorganization and recruitment, despite perturbation by chemical stimulation. Our findings will help elucidate the mechanisms of FN biosynthesis and organization by furthering the link of the role of mechanics with FN.

Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology.

Mechanotransduction of sensory neurons is of great interest to the scientific community, especially in areas such as pain, neurobiology, cardiovascular homeostasis and mechanobiology. We describe a method to investigate stretch-activated mechanotransduction in sensory nerves through subcellular stimulation. The method imposes localized mechanical stimulation through indentation of an elastomeric substrate and combines this mechanical stimulation with whole-cell patch clamp recording of the electrical response to single-nerve stretching. One significant advantage here is that the neurites are stretched with limited physical contact beyond their attachment to the polymer. When we imposed specific mechanical stimulation through the substrate, the stretched neurite fired and an action potential response was recorded. In addition, complementary protocols to control the molecules at the cell-substrate interface are presented. These techniques provide an opportunity to probe neurosensory mechanotransduction with a defined substrate, whose physical and molecular context can be modified to mimic physiologically relevant conditions. The entire process from fabrication to cellular recording takes 5 to 6 d.

Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate students.

We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students' understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates.

Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches.

The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell-material interactions.

Nanoscale intracellular organization and functional architecture mediating cellular behavior.

Cells function based on a complex set of interactions that control pathways resulting in ultimate cell fates including proliferation, differentiation, and apoptosis. The inter-workings of this immensely dense network of intracellular molecules are influenced by more than random protein and nucleic acid distribution where their interactions culminate in distinct cellular function. By probing the design of these biological systems from an engineering perspective, researchers can gain great insight that will aid in building and utilizing systems that are on this size scale where traditional large-scale rules may fail to apply. The organized interaction and gradient distribution in intracellular space imply a structural architecture that modulates cellular processes by influencing biochemical interactions including transport and binding-reactions. One significant structure that plays a role in this modulation is the cell cytoskeleton. Here, we discuss the cytoskeleton as a central and integrating functional structure in influencing cell processes and we describe technology useful for probing this structure. We explain the nanometer scale science of cytoskeletal structure with respect to intracellular organization, mechanotransduction, cytoskeletal-associated proteins, and motor molecules, as well as nano- and microtechnologies that are applicable for experimental studies of the cytoskeleton. This biological architecture of the cytoskeleton influences molecular, cellular, and physiological processes through structured multimodular and hierarchical principles centered on these functional filaments. Through investigating these organic systems that have evolved over billions of years, understanding in biology, engineering, and nanometer-scaled science will be advanced.

Characterization of an actin-binding site within the talin FERM domain.

Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles.

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.