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.T he biochemical response of cells to external mechanical stimulation has generated tremendous interest over the past decade. Of particular interest are the causative contributions of mechanotransductive transmembrane integrin molecules that link the extracellular matrix (ECM) to the cell interior and play a role in physiological response and molecular signaling within the cell. Recently, numerous exciting discoveries have evolved from these studies including the role of mechanotransduction in vascular physiology and atherogenesis, Src activation (Src phosphorylation/ activation under local mechanical stimulation), and the effect of matrix stiffness on stem cell differentiation (1-3). Mechanical stimulation has been shown to produce a wide range of cellular responses including the proteomic activation of the mitogenactivated protein kinase (MAPK) pathways in extracellular signalregulated kinases (ERKs), alterations of genomic expression profiles, and control of cell morphology, differentiation, and proliferation (4, 5). However, such research has focused primarily on the integrins as the mechanical signal initiator/transmembrane protein. Although other strain-sensitive proteins, such as mechanosensitive ion channels and protein kinase C, are known to be altered by mechanical stimulation, these responses are thought to occur downstream or be directly linked to the transmembrane proteininitiating...

Objectives/Hypothesis-The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of BrdU, a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA Synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using EdU as a thymidine analog and a non-antibody azide/alkyne reaction between the EdU and the fluorescent probe. We propose that EdU is as effective as BrdU but without the requirement for harsh denaturation or the use of antibodies for detection.Study Design-Two week-old chicks received a single gentamicin injection followed by a single EdU injection 72h later. Cochleae were extracted 4-8h later, fixed, and processed for fluorescent detection of EdU.Methods-Cochleae were processed for detection of incorporated EdU using the Click-iT™ Imaging Kit (Invitrogen) and co-labeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy.Results-Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8h following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions-TheEdU method is as effective as BrdU without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the non-antibody EdU system allows more flexibility by enabling co-labeling with multiple antibodies to other cellular proteins involved in regeneration.

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