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Biomedical subjects

P Kouklis

Publications and source records attributed to P Kouklis.

6 recordsLinked to original sources
PubMed2001

Ca(2+) signalling and PKCalpha activate increased endothelial permeability by disassembly of VE-cadherin junctions.

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Albumins↗
PubMed2000

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly.

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.

Adherens Junctions↗
PubMed1998

Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage.

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell-cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous -/- mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (-/-) embryos, the paucity of desmosomal cell-cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.

Animals↗
PubMed1996

An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments.

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Actin Cytoskeleton↗
PubMed1993

The roles of the rod end and the tail in vimentin IF assembly and IF network formation.

Using mutagenesis, we investigated the importance of two vimentin domains: (a) a highly conserved segment near the carboxy end of the alpha-helical rod, and (b) the tail, with which the rod end is known to interact. As judged by in vitro filament assembly and expression in transiently transfected cells lacking an endogenous vimentin network, the rod-tail interaction is not essential for 10 nm filament structure in vitro or for formation of fibrous arrays in culture. However, when mutated, amino acid residues within the rod and the tail segments can cause perturbations in IF assembly and in IF network formation. Finally, our studies show that the vimentin tail seems to play a role both in thermodynamically stabilizing IF structure in vitro and in establishing proper IF networks in vivo.

Amino Acid Sequence↗