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PubMed2002

Myotonic dystrophy protein kinase of the cardiac muscle: evaluation using an immunochemical approach.

Myotonic dystrophy (DM) is an inherited multisystem disorder characterized by the presence of a high polymorphic expansion of trinucleotide (CTG) repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. However, the role of myotonic dystrophy protein kinase (DMPK) has yet to be elucidated. Studies aimed to discover possible physiological targets of DMPK indicated several subcellular localization sites, such as neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum in the skeletal muscle and intercalated discs in the cardiac muscle. Here, we extend our previous observations on the localization of DMPK at gap junction (GJ) level in the heart, taking advantage of the polyclonal peptide-specific anti-DMPK antibodies raised against two different domains of the protein. DMPK was detected by immunofluorescence at the intercalated disc level by both antibodies. Double immunofluorescence staining experiments performed with each anti-DMPK and anti-connexin43 showed colocalization of the two antigens. Immunoblot analysis of partially purified GJs showed co-sedimentation of DMPK and connexin43. We conclude that GJs are a genuine localization site of DMPK. Given the known regulation exerted by protein kinases on assembly, trafficking, gating, and disassembly of connexins, such a localization may be relevant to the functional role of connexins. DM is the most common muscular dystrophy in adults, and is known by the cardiac involvement that is a common feature in DM patients. Localization of DMPK at GJ in relation to DM is also briefly discussed.

Animals↗
PubMed1999

Excitatory granule cells of the dentate gyrus exhibit a double inhibitory neurochemical content after intrahippocampal administration of kainate in adult mice.

We have previously demonstrated that, in the adult mouse, injection of kainate/AMPA receptors agonists into the dorsal hippocampus induces major structural modifications of the dentate gyrus granule cells. Such changes are mediated by the brain-derived neurotrophic factor (BDNF). Considering previous involvements of BDNF in activity-linked regulations of hippocampal neuronal phenotype, changes of neurochemical contents were further investigated. It is shown that excitatory granule cells rapidly acquire a strong immunoreactivity for the inhibitory neurotransmitters GABA and neuropeptide-Y, with different patterns for both molecules. GABA immunoreactivity appeared first in mossy fibers, before extending to cell bodies and dendrites. Analysis of glutamic acid decarboxylase revealed slight increases in mossy fibers and no somatic labeling. In contrast to GABA, neuropeptide-Y labeling was observed first in granule cell soma and then in mossy fibers, with a centrifugal gradient. All labelings were transient, but slight amounts of GABA and NPY were kept in some cell bodies for at least 6 months. Confocal microscope analysis of double GABA/NPY labelings revealed colocalization of both mediators in the same neurons. The specificity of kainate-linked changes was suggested by lack of immunoreactivity for somatostatin. These results show that the capacities of mature granule cells to adapt environmental modifications can concern neurochemical contents, by synthesis and/or uptake of specific molecules. The fact that adaptive changes are rapid and transient suggests a direct response to kainate, in order to limit its potentially deleterious effects. Colocalization of GABA and neuropeptide-Y indicates that the dentate gyrus granule cells can use several pathways to this aim.

Age Factors↗
PubMed1997

Intratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phosphatidylcholine/cholesterol liposomes.

PURPOSE: A pharmacological evaluation of an egg phosphatidylcholine/cholesterol (55:45 mole ratio, EPC/Chol) liposome doxorubicin formulation was carried out. The objective was to define liposomal lipid and drug distribution within sites of tumor growth following intravenous (i.v.) administration to female BDF1 mice bearing either Lewis lung carcinoma, B16/BL6 melanoma, or L1210 ascitic tumors. METHODS: Mice were injected i.v. with EPC/Chol liposomal doxorubicin, and plasma and tumor levels of lipid and drug were determined 1, 4 and 24 h late with radiolabeled lipid and fluorimetry or fluorescence microscopy, respectively. In addition, single-cell suspensions of the Lewis lung and B16/BL6 tumors were prepared and the presence of macrophages was determined with an FITC-labeled rat antimouse CD11b (MAC-1) antibody. RESULTS: For mice bearing the Lewis lung solid tumors, there was a time-dependent accumulation of liposomal lipid, with a plateau of approximately 500 micrograms lipid/g tumor at 48 h. In contrast, the apparent plateau (microgram doxorubicin/g tumor) for doxorubicin was achieved at 1 h and remained constant over a 72-h time course. In comparison with free drug administered at the maximum tolerated dose (MTD, 20 mg/kg) doxorubicin levels in tumors were two- to threefold greater when the drug was administered in liposomal form. The increase in drug delivery was comparable for both solid tumors. With animals bearing the L1210 ascitic tumor, drug exposure was as much as ten times greater (in comparison with free drug) when doxorubicin was administered in liposomes. An evaluation of single-cell suspensions prepared from the two solid tumors suggested that more than 98% of the tumor-associated drug and liposomal lipid was not tumor cell-associated. Histological studies with the Lewis lung carcinoma, however, revealed that a proportion of the drug did colocalize with tumor-associated macrophages. Analysis of cells obtained from mice bearing ascitic tumors showed that more than 80% of the cell-associated drug could be removed by procedures designed to remove adherent cells. CONCLUSION: The results summarized here suggest drug concentrations within a solid tumor, such as the Lewis lung carcinoma, are constant over time when the drug is given in a "leaky" EPC/Chol formulation. The results also suggest that liposomal lipid within sites of tumor growth is primarily localized within the interstitial spaces or tumor-associated macrophages.

Animals↗
PubMed2004

Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity.

The Cyclin D3 protein is a member of the D-type cyclins. Besides serving as cell cycle regulators, D-type cyclins have been reported to be able to interact with several transcription factors and modulate their transcriptional activations. Here we report that human activating transcription factor 5 (hATF5) is a new interacting partner of Cyclin D3. The interaction was confirmed by in vivo coimmunoprecipitation and in vitro binding analysis. Neither interaction between Cyclin D1 and hATF5 nor interaction between Cyclin D2 and hATF5 was observed. Confocal microscopy analysis showed that Cyclin D3 could colocalize with hATF5 in the nuclear region. Cyclin D3 could potentiate hATF5 transcriptional activity independently of its Cdk4 partner. But Cyclin D1 and Cyclin D2 had no effect on hATF5 transcriptional activity. These data provide a new clue to understand the new role of Cyclin D3 as a transcriptional regulator.

Animals↗
PubMed2006

Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells.

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.

Adrenal Medulla↗
PubMed2006

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia.

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.

Animals↗
PubMed2006

Sumoylation of Rta of Epstein-Barr virus is preferentially enhanced by PIASxbeta.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the viral lytic cycle. This study identifies PIASxalpha and PIASxbeta as binding partners of Rta in a yeast two-hybrid screen and demonstrates the binding of Rta to PIASxalpha and PIASxbeta in vitro by GST pull-down analysis. Coimmunoprecipitation experiments and indirect immunofluorescence analysis show that Rta interacts and colocalizes with PIASxalpha and PIASxbeta in the nucleus. These interactions seem to enhance Rta sumoylation as transfecting plasmids expressing PIASxalpha, PIASxbeta, Ubc9, or SUMO-1 increase the capacity of Rta to transactivate a promoter that contains an Rta-response element and the promoters of p21 and BNLF1 in transient transfection assay. This study also finds that Rta sumoylation is preferentially enhanced by PIASxbeta, which could be attributed to the fact that PIASxbeta, compared to PIASxalpha, has a strong affinity to Rta, suggesting that affinity of a SUMO E3 ligase to its target protein influences the function of protein sumoylation.

Cell Nucleus↗
PubMed2005

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules.

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.

Animals↗
PubMed2000

Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis.

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein-protein interaction may contribute to the regulation of LTC4 production.

Animals↗
PubMed2001

Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses.

Influenza virus assembly and morphogenesis require transport of viral components to the assembly site at the apical plasma membrane of polarized epithelial cells and interaction among the viral components. In this report we have discussed the apical determinants present in the transmembrane domain (TMD) of influenza virus hemagglutinin (HA) and neuraminidase (NA), and the interaction of M1 with influenza virus HA and NA. Earlier studies have shown that the NA and HA TMDs possess determinant(s) for apical sorting and raft-association (Kundu et al., 1996. J. Virol 70, 6508-6515; Lin et al., 1998. J. Cell Biol. 142, 51-57). Analysis of chimeric constructs between NA and TR (human transferring receptor) TMDs and the mutations in the NA and HA TMD sequences showed that the COOH terminus of the NA TMD and NH(2) terminus of the HA TMD encompassing the exoplasmic leaflet of the lipid bilayers were significantly involved in lipid raft-association and that apical determinants were not discrete sequences but rather dispersed within the TMD of HA and NA. These analyses also showed that although both signals for apical sorting and raft-association resided in the NA TMD, they were not identical and varied independently. Interactions of M1 protein with HA or NA, the influenza virus envelope glycoproteins, were investigated by TX-100 detergent treatment of membrane fractions and floatation in sucrose gradients. Results from these analyses showed that the interaction of M1 with mature HA and NA, which associated with the detergent-resistant lipid rafts caused an increased detergent-resistance of the membrane-bound M1 and that M1 interacted with HA and NA both in influenza virus-infected cells as well as in recombinant vaccinia virus-infected cells coexpressing M1 with HA and/or NA. Furthermore, both the cytoplasmic tail and the TMD of HA caused an increased detergent-resistance of the membrane-bound M1 supporting their interaction with M1. Immunofluorescence analysis by confocal microscopy also showed colocalization supporting the interaction of M1 with HA and NA at the cell surface and during exocytic transport both in influenza virus-infected cells as well as in coexpressing cells.

Amino Acid Sequence↗
PubMed2004

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

Amino Acid Sequence↗
PubMed1999

A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein, the subunit c of ATP synthase.

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.

Aminopeptidases↗
PubMed1996

Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alpha-actinin.

Intercellular adhesion molecule-2 (ICAM-2) functions as a ligand for lymphocyte function-associated antigen-1 (LFA-1) and is involved in leukocyte adhesion. We studied intracellular associations of ICAM-2 using a peptide encompassing the cytoplasmic amino acids 231-254 as an affinity matrix. Among the proteins from placental lysates that bound to the peptide was alpha-actinin as demonstrated by immunoblotting. Purified, 125I-labeled alpha-actinin also bound to the peptide. Confocal microscopic analysis of Eahy926 cells demonstrated a colocalization of ICAM-2 and alpha-actinin. Of overlapping octapeptides covering the entire ICAM-2 cytoplasmic amino acids, ICAM-2241-248 bound alpha-actinin most avidly and effectively competed with the longer cytoplasmic peptide for binding. The site of interaction in alpha-actinin was studied using bacterially expressed alpha-actinin fusion proteins. Several constructs covering nonoverlapping regions of alpha-actinin bound to the ICAM-2 cytoplasmic peptide suggesting that multiple regions in alpha-actinin can mediate the interaction. These results, together with previously demonstrated interactions between alpha-actinin and the adhesion proteins ICAM-1, L-selectin, beta1- and beta2-integrins emphasize the role of alpha-actinin as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton.

Actinin↗
PubMed2001

The Slp homology domain of synaptotagmin-like proteins 1-4 and Slac2 functions as a novel Rab27A binding domain.

rab27A, which encodes a small GTP-binding protein, was recently identified as a gene in which mutations caused human hemophagocytic syndrome (Griscelli syndrome) and ashen mice, which exhibit defects in melanosome transport as well as in regulated granule exocytosis in cytotoxic T lymphocytes. However, little is known about the molecular mechanism of Rab27A-dependent membrane trafficking or the specific effector molecules of Rab27A. In this study, we discovered that the Slp (synaptotagmin-like protein) homology domain (SHD) of Slp1--3 and Slac2-a/b specifically and directly binds the GTP-bound form of Rab27A both in vitro and in intact cells but not of the other Rabs tested (Rab1, Rab2, Rab3A, Rab4, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). Immunocytochemical analysis revealed that Slp2 (or Slp1) colocalized with Rab27A in the melanosomes of melanoma cells. Slp2 and Rab27A were distributed to the periphery of the cells (especially at the dendritic tips) in the wild-type melanoma cells, whereas they accumulated in the perinuclear region in the melanosome transport-defective cells (S91/Cloudman). These results strongly indicated that the SHD of Slp1--3 and Slac2 functions as an in vivo Rab27A binding domain.

Amino Acid Sequence↗
PubMed2003

Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis.

Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.

Animals↗
PubMed2000

Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution.

The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

Cell Cycle↗
PubMed2005

Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels.

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body. Most, but not all, of these genes are induced in functionally mature CD80(hi) mTECs. Although the autoimmune regulator (Aire) is responsible for inducing a large portion of this gene pool, numerous tissue-restricted genes are also up-regulated in mature mTECs in the absence of Aire. Promiscuously expressed genes tend to colocalize in clusters in the genome. Analysis of a particular gene locus revealed expression of clustered genes to be contiguous within such a cluster and to encompass both Aire-dependent and -independent genes. A role for epigenetic regulation is furthermore implied by the selective loss of imprinting of the insulin-like growth factor 2 gene in mTECs. Our data document a remarkable cellular and molecular specialization of the thymic stroma in order to mimic the transcriptome of multiple peripheral tissues and, thus, maximize the scope of central self-tolerance.

Animals↗
PubMed2000

The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis.

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.

Actins↗