The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line

1994 ◽  
Vol 107 (10) ◽  
pp. 2909-2918 ◽  
Author(s):  
L. Baricault ◽  
B. de Nechaud ◽  
C. Sapin ◽  
P. Codogno ◽  
P. Denoulet ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Phosphorylation Site ◽  
Human Colon ◽  
Cell System ◽  
Cytoskeletal Proteins ◽  
Cell Periphery ◽  
Type I ◽  
Cellular Functions ◽  
Morphological Alterations ◽  
Continuous Presence

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions.

scholarly journals The Spectraplakin Short Stop Is an Actin–Microtubule Cross-Linker That Contributes to Organization of the Microtubule Network

2010 ◽  
Vol 21 (10) ◽  
pp. 1714-1724 ◽  
Author(s):  
Derek A. Applewhite ◽  
Kyle D. Grode ◽  
Darby Keller ◽  
Alireza Dehghani Zadeh ◽  
Kevin C. Slep ◽  
...  
Keyword(s):  
Cross Talk ◽  
Function Analysis ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Cross Linking ◽  
Cell Periphery ◽  
Cellular Functions ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Cross Linker

The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk.

scholarly journals The Syndrome of Carnitine Deficiency: Morphological and Metabolic Correlations in Two Cases

1978 ◽  
Vol 5 (2) ◽  
pp. 205-213 ◽  
Author(s):  
G. Scarlato ◽  
G. Pellegrini ◽  
C. Cerri ◽  
G. Meola ◽  
A. Veicsteinas
Keyword(s):  
Lactic Acid ◽  
Muscle Tissue ◽  
Clinical Manifestations ◽  
Carnitine Deficiency ◽  
Type I ◽  
Work Intensity ◽  
Morphological Alterations ◽  
Ultrastructural Studies ◽  
First Case ◽  
Type I Fibers

SUMMARY:Two cases of systemic carnitine deficiency are described. In both patients, carnitine concentration was lower than normal in serum and muscle tissue. In the first case, the illness began at age 35; the clinical manifestations were only muscular. In the second case, the illness began in childhood; there were intermittent episodes of hepatic enlargement and coma. An excessive lipid content was present in muscle tissue, especially in type I fibers, of both cases, and in the liver of the second patient. Ultrastructural studies of muscle tissue revealed important changes of mitochondria.During muscular exercise, aerobic and anaerobic metabolism were in vestigated. For a given relative work intensity, these patients showed abnormally high blood lactic acid concentration and lactic acid/pyruvic acid ratios. These data, together with the morphological alterations observed in mitochondria, suggest an impaired function of the respiratory chain, leading to a shift of the red/ox potential of the tissue towards a non reduced state.

scholarly journals Skimmer CID‒ion trap mass spectrometry of phosphotyrosine‒containing peptides

2004 ◽  
Vol 18 (3) ◽  
pp. 441-451
Author(s):  
Melissa D. Zolodz ◽  
Karl V. Wood
Keyword(s):  
Mass Spectrometry ◽  
Ion Trap ◽  
Phosphorylation Site ◽  
Ion Trap Mass Spectrometry ◽  
Cellular Functions ◽  
Factors Affecting ◽  
Parent Protein ◽  
Phosphorylated Peptides ◽  
Phosphorylated Peptide ◽  
Analytical Tools

Proteomic analysis is becoming a popular field in science. Analysis of protein modifications is useful in deciphering cellular functions and errors in pathways that can result in disease. There has been increased interest in the phosphotyrosine proteome. Due to the difficulty in finding the location of the tyrosine phosphorylation site in the tyrosine phosphorylated peptide or even to verify that the parent protein is a phosphotyrosyl‒protein, new analytical tools are being developed. The phosphotyrosine immonium ion can be produced via skimmer CID for detection via ion trap mass spectrometry and is a useful marker for the indication of the presence of a phosphotyrosine residue. Skimmer CID analysis can also be used to differentiate phosphotyrosine‒containing peptides from other phosphorylated peptides. In this study, phosphotyrosine‒containing peptides were analyzed by skimmer CID in an ion trap mass spectrometer. The factors affecting the signal abundance of the phosphotyrosine immonium ion were investigated.

scholarly journals Localization and Interactions of Teichoic Acid Synthetic Enzymes in Bacillus subtilis

2007 ◽  
Vol 190 (5) ◽  
pp. 1812-1821 ◽  
Author(s):  
Alex Formstone ◽  
Rut Carballido-López ◽  
Philippe Noirot ◽  
Jeffery Errington ◽  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Cell Shape ◽  
Spatial Organization ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Teichoic Acid ◽  
Cytoskeletal Proteins ◽  
Thick Wall ◽  
Wall Teichoic Acid ◽  
Critical Function

ABSTRACT The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.

scholarly journals Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

2014 ◽  
Vol 207 (3) ◽  
pp. 407-418 ◽  
Author(s):  
Sara Löchte ◽  
Sharon Waichman ◽  
Oliver Beutel ◽  
Changjiang You ◽  
Jacob Piehler
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Polymer Brush ◽  
Effector Proteins ◽  
Live Cells ◽  
Type I ◽  
Rgd Peptides ◽  
Single Molecule Level ◽  
Cell Micropatterning

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts

1998 ◽  
Vol 111 (13) ◽  
pp. 1897-1907 ◽  
Author(s):  
B. Eckes ◽  
D. Dogic ◽  
E. Colucci-Guyon ◽  
N. Wang ◽  
A. Maniotis ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Mechanical Stability ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Cellular Functions ◽  
Primary Fibroblasts ◽  
In Vitro Systems ◽  
Filament Network ◽  
Collagen Lattices

Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

scholarly journals Distinct segregation patterns of yeast cell-peripheral proteins uncovered by a method for protein segregatome analysis

2019 ◽  
Vol 116 (18) ◽  
pp. 8909-8918 ◽  
Author(s):  
Shinju Sugiyama ◽  
Motomasa Tanaka
Keyword(s):  
Wall Stress ◽  
Cell Periphery ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Binding Domains ◽  
Peripheral Proteins ◽  
Flow Through ◽  
Membrane Spanning ◽  
Mother Cells ◽  
Er Membrane

Protein segregation contributes to various cellular processes such as polarization, differentiation, and aging. However, the difficulty in global determination of protein segregation hampers our understanding of its mechanisms and physiological roles. Here, by developing a quantitative proteomics technique, we globally monitored segregation of preexisting and newly synthesized proteins during cell division of budding yeast, and identified crucial domains that determine the segregation of cell-peripheral proteins. Remarkably, the proteomic and subsequent microscopic analyses demonstrated that the flow through the bud neck of the proteins that harbor both endoplasmic reticulum (ER) membrane-spanning and plasma membrane (PM)-binding domains is not restricted by the previously suggested ER membrane or PM diffusion barriers but by septin-mediated partitioning of the PM-associated ER (pmaER). Furthermore, the proteomic analysis revealed that although the PM-spanning t-SNARE Sso2 was retained in mother cells, its paralog Sso1 unexpectedly showed symmetric localization. We found that the transport of Sso1 to buds was required for enhancement of polarized cell growth and resistance to cell-wall stress. Taken together, these data resolve long-standing questions about septin-mediated compartmentalization of the cell periphery, and provide new mechanistic insights into the segregation of cell-periphery proteins and their cellular functions.

scholarly journals Inositol trisphosphate analogues selective for types I and II inositol trisphosphate receptors exert differential effects on vasopressin-stimulated Ca2+ inflow and Ca2+ release from intracellular stores in rat hepatocytes

2004 ◽  
Vol 381 (2) ◽  
pp. 519-526 ◽  
Author(s):  
Roland B. GREGORY ◽  
Rachael HUGHES ◽  
Andrew M. RILEY ◽  
Barry V. L. POTTER ◽  
Robert A. WILCOX ◽  
...  
Keyword(s):  
Single Cells ◽  
Inositol Trisphosphate ◽  
Rat Hepatocytes ◽  
Small Region ◽  
Cell Periphery ◽  
Type I ◽  
Response Curves ◽  
Maximal Stimulation ◽  
Adenophostin A ◽  
Stimulation Of

Previous studies have shown that adenophostin A is a potent initiator of the activation of SOCs (store-operated Ca2+ channels) in rat hepatocytes, and have suggested that, of the two subtypes of Ins(1,4,5)P3 receptor predominantly present in rat hepatocytes [Ins(1,4,5)P3R1 (type I receptor) and Ins(1,4,5)P3R2 (type II receptor)], Ins(1,4,5)P3R1s are required for SOC activation. We compared the abilities of Ins(1,4,6)P3 [with higher apparent affinity for Ins(1,4,5)P3R1] and Ins(1,3,6)P3 and Ins(1,2,4,5)P4 [with higher apparent affinities for Ins(1,4,5)P3R2] to activate SOCs. The Ins(1,4,5)P3 analogues were microinjected into single cells together with fura 2, and dose–response curves for the activation of Ca2+ inflow and Ca2+ release from intracellular stores obtained for each analogue. The concentration of Ins(1,4,6)P3 which gave half-maximal stimulation of Ca2+ inflow was substantially lower than that which gave half-maximal stimulation of Ca2+ release. By contrast, for Ins(1,3,6)P3 and Ins(1,2,4,5)P3, the concentration which gave half-maximal stimulation of Ca2+ inflow was substantially higher than that which gave half-maximal stimulation of Ca2+ release. The distribution of Ins(1,4,5)P3R1 and Ins(1,4,5)P3R2 in rat hepatocytes cultured under the same conditions as those employed for the measurement of Ca2+ inflow and release was determined by immunofluorescence. Ins(1,4,5)-P3R1s were found predominantly at the cell periphery, whereas Ins(1,4,5)P3R2s were found at the cell periphery, the cell interior and nucleus. It is concluded that the idea that a small region of the endoplasmic reticulum enriched in Ins(1,4,5)P3R1 is required for the activation of SOCs is consistent with the present results for hepatocytes.

scholarly journals Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

1998 ◽  
Vol 9 (5) ◽  
pp. 993-1006 ◽  
Author(s):  
Shigeko Yamashiro ◽  
Yoshihiko Yamakita ◽  
Shoichiro Ono ◽  
Fumio Matsumura
Keyword(s):  
Epithelial Cells ◽  
Cell Motility ◽  
Transformed Cells ◽  
Boyden Chamber ◽  
Cell Periphery ◽  
Migration Activity ◽  
Normal Cells ◽  
Morphological Alterations ◽  
Membrane Protrusions ◽  
Antigen Presenting

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.

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