Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation

Abstract

Although much is known about the molecules involved in extracellular Ca 2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca 2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein α 5 -integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca 2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

“…Using indirect immunofluorescence, α5-integrin was detected in fibrillar adhesions at the terminations of stress fibers in both control and 24-h EGTA-treated C2C12 cells. Besides, a dense α5-integrin labeling was seen inside most treated cells, indicating a possible internalization of integrin after EGTA exposure (12). The effects of calcium depletion on desmin distribution observed in the present study may have been initiated by various cell surface components that require extracellular Ca 2+ , such as α5-integrin, which in turn can influence intracellular signaling pathways.…”

“…One major Ca 2+ -dependent transmembrane protein found in the sarcolemma of myoblasts is integrin. Previous work from our group showed a change in the distribution of α5-integrin adhesion sites in C2C12 cells after EGTA treatment (12). Using indirect immunofluorescence, α5-integrin was detected in fibrillar adhesions at the terminations of stress fibers in both control and 24-h EGTA-treated C2C12 cells.…”

“…cDNA was synthesized from 3 µg total RNA. RNA was primed with 0.5 µg oligo-(dT) [12][13][14][15][16][17][18] primer (reaction volume: 20 µl). After RNA was denatured (10 min at 70ºC) and cooled on ice, the following reagents were mixed and then added: 10 mM of each deoxynucleoside triphosphate (dNTP), 6 µl 5x reverse transcription (RT) buffer, 0.1 mM DTT, and 200 U superscript RT (reaction volume: 30 µl).…”

“…Previous work from our group suggested a role for extracellular Ca 2+ in the regulation of the spatial distribution of microfilaments, microtubules, intermediate filaments, and adhesion sites in myoblasts (12). The present investigation was undertaken to study the effects of extracellular Ca 2+ withdrawal on cell shape and on the expression and distribution of desmin in myoblasts grown in culture.…”

Autoantibodies to Ca2+ binding protein Calnuc is a potential marker in colon cancer detection

“…The cytoplasmic pool is more likely to regulate other proteins through direct interaction, as with other Ca 2+ -binding proteins such as calmodulin, troponin C, and recoverin located in the cytoplasm (33). It was observed that cell shape, adhesion, and motility were affected by Caregulated pathways, which depended on Ca 2+ -binding proteins (34). Previous study has reported that one ER Ca 2+ binding protein calreticulin, which shows significant sequence homology to Calnuc, affects cell adhesion via the regulation of expression of proteins which is important in adhesion, as well as via its effects on intracellular signaling pathways (35)(36)(37)(38).…”