and protein synthesis by modulation of nuclear ..
Brabletz and collaborators (20) showed that nuclear ..
We report a method for both precisely controlling cell and nuclear shape and measuring in situ mRNA expression in an effort to help elaborate these mechanisms. Microfabrication and fluidic techniques have been used to control cell adhesion and spreading on the μm scale and have proven useful for asking basic questions in cell biology (, , , –). Although previous studies have used surfaces with spatially resolved chemistry to study cell shape/function relationships and their effect on protein synthesis (, ), studies examining in situ gene expression (mRNA) and protein synthesis as a function of the cell projected area and nuclear shape have not been published. Therefore, the methods developed in this work were designed to assess cell and nuclear shape-dependent changes in gene expression and protein synthesis of individual cells. An interpenetrating polymer network of poly(acrylamide) and polyethylene glycol [p(AAm-co-EG)] grafted to silane-derivatized glass was used as a nonadhesive surface chemistry amenable to the thermal cycling of PCR (). Combining photolithographic and photopolymerization techniques, we created islands of adhesive surface chemistry [an amine-terminated silane that preferentially adsorbs vitronectin ()] surrounded by the p(AAm-co-EG) interpenetrating polymer network. A range of geometric shapes and sizes were created to impart different mechanical environments within different cells maintained on the same culture surface. Because of our interest in bone, we patterned primary bone-derived cells from rat calvaria and probed for type I collagen (CollI) synthesis and osteocalcin (OC) mRNA expression to determine whether constraining the projected area and nuclear shape of a bone-derived cell altered the time required for differentiation into the osteoblast phenotype. We chose CollI and OC because they are markers of osteoblast differentiation, and NMPs (e.g., Cbfa1, a transcriptional activator of osteoblast differentiation) have been shown to bind to the promoter region of genes for both OC and CollI ().
Modulation of gene expression and collagen ..
Cell morphology has a profound effect on a range of cellular events, such as proliferation (–), differentiation (–), cytoskeletal organization (), and presumably gene expression. Changes to the cytoskeleton lead to altered stress levels imparted on the nucleus (–) and could affect organelle and DNA organization and distribution, ultimately altering cell function. For example, the rate of albumin secretion from hepatocytes can be altered by constraining cell size on patterned culture surfaces (). In human epidermal kareatinocytes cell shape can modulate between terminal differentiation and proliferation (). Furthermore, cells can be forced to enter the apoptotic cascade when the area on which the cell is allowed to spread is constrained (). One proposed mechanism for the transduction of cell shape information into gene expression is through mechanical forces transmitted by means of the direct link of the cytoskeleton to the nucleus (, ), and in particular to nuclear matrix proteins (NMPs) such as NMP-1 and NMP-2 (). These architectural transcription factors, which are components of the nuclear scaffold, induce changes in DNA supercoiling and can interact directly with gene promoter sequences (). This interaction between the nuclear architecture and regulation of transcription or DNA topography raises the question of whether gross deformation of the cell, and hence its nucleus, can modulate the NMP/DNA interactions and gene expression. However, the details in the cascade of mechanical events involving cell morphology, cytoskeletal organization, intracellular signaling, nuclear shape, nuclear matrix organization, promoter geometry, and gene expression are poorly understood.