Constraint-based reasoning in cell biology: on the explanatory role of context.

Cell biologists, including those seeking molecular mechanistic explanations of cellular phenomena, frequently rely on experimental strategies focused on identifying the cellular context relevant to their investigations. We suggest that such practices can be understood as a guided decomposition strategy, where molecular explanations of phenomena are defined in relation to natural contextual (cell) boundaries. This "top-down" strategy contrasts with "bottom-up" reductionist approaches where well-defined molecular structures and activities are orphaned by their displacement from actual biological functions.

BAMBI is a novel HIF1-dependent modulator of TGFβ-mediated disruption of cell polarity during hypoxia.

Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of ∼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation.

Laminins in Epithelial Cell Polarization: Old Questions in Search of New Answers.

Laminin, a basement membrane protein discovered in 1979, was shortly thereafter implicated in the polarization of epithelial cells in both mammals and a variety of lower organisms. To transduce a spatial cue to the intrinsic polarization machinery, laminin must polymerize into a dense network that forms the foundation of the basement membrane. Evidence suggests that activation of the small GTPase Rac1 by β1-integrins mobilizes laminin-binding integrins and dystroglycan to consolidate formation of the laminin network and initiate rearrangements of both the actin and microtubule cytoskeleton to help establish the apicobasal axis.

The secretory pathway at 50: a golden anniversary for some momentous grains of silver.

The secretory pathway along which newly synthesized secretory and membrane proteins traffic through the cell was revealed in two articles published 50 years ago. This discovery was the culmination of decades of effort to unite the power of biochemical and morphological methodologies in order to elucidate the dynamic nature of the cell's biosynthetic machinery. The secretory pathway remains a central paradigm of modern cell biology.

The Heuristic of Form: Mitochondrial Morphology and the Explanation of Oxidative Phosphorylation.

In the 1950s and 1960s, the search for the mechanism of oxidative phosphorylation by biochemists paralleled the description of mitochondrial form by George Palade and Fritiof Sjöstrand using electron microscopy. This paper explores the extent to which biochemists studying oxidative phosphorylation took mitochondrial form into account in the formulation of hypotheses, design of experiments, and interpretation of results. By examining experimental approaches employed by the biochemists studying oxidative phosphorylation, and their interactions with Palade, I suggest that use of mitochondrial form as a guide to experimentation and interpretation varied considerably among investigators.

αV-integrins are required for mechanotransduction in MDCK epithelial cells.

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood.

Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference.

Spatial expression of the genome: the signal hypothesis at forty.

The signal hypothesis, formulated by Günter Blobel and David Sabatini in 1971, and elaborated by Blobel and his colleagues between 1975 and 1980, fundamentally expanded our view of cells by introducing the concept of topogenic signals. Cells were no longer just morphological entities with compartmentalized biochemical functions; they were now active participants in the creation and perpetuation of their own form and identity, the decoders of linear genetic information into three dimensions.

Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed.

Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control).

Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway.

In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells.

The protein kinase A pathway contributes to Hg2+-induced alterations in phosphorylation and subcellular distribution of occludin associated with increased tight junction permeability of salivary epithelial cell monolayers.

Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction.

Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair.

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects.

Regulated synthesis and functions of laminin 5 in polarized madin-darby canine kidney epithelial cells.

Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical-basal polarization of Madin-Darby canine kidney (MDCK) cells.

Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia.

In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl(-) secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl(-) secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC.

Beta1-integrin orients epithelial polarity via Rac1 and laminin.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures.

Differential subcellular targeting of PKC-epsilon in response to pharmacological or ischemic stimuli in intestinal epithelia.

Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling.

Regulation of MDCK cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion.

The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable.

Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis.

Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin.

Induction of a laminin isoform and alpha(3)beta(1)-integrin in renal ischemic injury and repair in vivo.

Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes.