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P Gunning

Publications and source records attributed to P Gunning.

At least 37 records · Page 2Linked to original sources
PubMed1997

Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones.

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

Actin Cytoskeleton↗
PubMed1997

Actin and tropomyosin isoforms in morphogenesis.

The major components of the actin microfilament system, actin and tropomyosin (Tm), are encoded by multigene families. There are at least 6 actin and over 20 Tm isoforms in mammals. The observation that isoforms are expressed in a tissue-specific manner has encouraged the hypothesis that they contribute to the formation of cell type-specific structures. Recent studies have indicated that certain specific isoforms do play unique structural roles. One nonmuscle actin isoform, beta, is implicated in the regulation of cell spreading and membrane organisation. The intracellular location of beta-actin mRNA has been shown to be regulated by growth factor stimulation of signal transduction pathways. Actin isoforms have also been shown to differ in their contractile properties in both muscle and non-muscle cells. Tropomyosins have been found to show isoform specific regulation in response to cell transformation. This has correlated with the view that some isoforms of tropomyosin promote filament stability whereas others are associated with more dynamic structures. Neuronal development and maturation are accompanied by dynamic spatial sorting of tropomyosin isoforms into different cellular compartments. It is now apparent that isoforms of these proteins perform different structural tasks. The challenge is now to link the significance of spatial sorting to the different physicochemical properties of these isoforms.

Actin Cytoskeleton↗
PubMed1997

Impact of alpha-skeletal actin but not alpha-cardiac actin on myoblast morphology.

Muscle differentiation involves a profound change in cell cytoarchitecture. This is accompanied by extensive isoform replacement in which the major non-muscle isoforms of the actin filament system are replaced by their muscle isoform counterparts. We have tested whether the sequential expression of the actin isoforms is functionally significant by precociously expressing the two striated muscle actins (alpha-skeletal and alpha-cardiac) in mouse myoblasts. The human alpha-skeletal and alpha-cardiac actin genes were transfected into mouse C2 myoblasts and clones expressing the human genes at the highest level were identified. Expression of the human alpha-skeletal actin gene was low with the highest mRNA level found to be 4% of that in adult human skeletal muscle. Clones expressing alpha-cardiac actin accumulated the mRNA up to 13% of the level of alpha-skeletal actin in adult human skeletal muscle. Despite the low level of alpha-skeletal actin expression, myoblasts transfected with this gene displayed a profound decrease in cell spreading. In contrast, alpha-cardiac actin had no impact on cell spreading. Neither alpha-skeletal actin nor alpha-cardiac actin had any impact on the total actin protein pool nor on the levels of the high molecular weight tropomyosins. The organisation of actin and tropomyosin into stress fibres was similar between transfected and control cells. We conclude that precocious expression of alpha-skeletal actin, but not alpha-cardiac actin, compromises myoblast morphology but not the ability of the cell to assemble stress-fibre-like structures.

Actins↗
PubMed1996

The molecular composition of neuronal microfilaments is spatially and temporally regulated.

The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm-5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, beta-actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of beta-actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta-actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and beta-actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation.

Actin Cytoskeleton↗
PubMed1995

Intracellular localization of tropomyosin mRNA and protein is associated with development of neuronal polarity.

Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.

Animals↗
PubMed1995

Impact of altered actin gene expression on vinculin, talin, cell spreading, and motility.

Previous studies have demonstrated a strong correlation between the expression of vinculin and the shape and motility of a cell (Rodriguez Fernandez et al., 1992a, b, 1993). This hypothesis was tested by comparing the expression of vinculin and talin with the motility of morphologically altered myoblasts. These mouse C2 myoblasts were previously generated by directly perturbing the cell cytoskeleton via the stable transfection of a mutant-form of the beta-actin gene (beta sm) and three different forms of the gamma-actin gene; gamma, gamma minus 3'UTR (gamma delta'UTR), and gamma minus intron III (gamma delta IVSIII) (Schevzov et al., 1992; Lloyd and Gunning, 1993). In the case of the beta sm and gamma-actin transfectants, a two-fold decrease in the cell surface area was coupled, as predicted, with a decrease in vinculin and talin expression. In contrast, the gamma delta IVSIII transfectants with a seven-fold decrease in the cell surface area showed an unpredicted slight increase in vinculin and talin expression and the gamma delta 3'-UTR transfectants with a slight increase in the cell surface area showed no changes in talin expression and a decrease in vinculin expression. We conclude that changes in actin gene expression alone can impact on the expression of vinculin and talin. Furthermore, we observed that these actin transfectants failed to show a consistent relationship between cell shape, motility, and the expression of vinculin. However, a relationship between talin and cell motility was found to exist, suggesting a role for talin in the establishment of focal contacts necessary for motility.

Actins↗
PubMed1994

Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell.

Cell motility is dependent upon the reorganization of the cellular cytoskeleton. Actin filaments form the major component of the cytoskeleton and respond rapidly to serum growth factors. We have previously shown that myoblasts sort the two cytoskeletal beta- and gamma-actin isoform mRNAs to different intracellular regions and that only beta-actin mRNA was associated with peripheral regions of cell motility (Hill, M.A. and P. Gunning. 1993. J. Cell Biol. 122: 825-832). We now show by in situ hybridization that 3T3 fibroblasts similarly sort actin isoform mRNAs and that peripheral beta-actin mRNA is regulated by serum. In the absence of serum, we could not detect beta-actin mRNA at the periphery. Addition of serum rapidly redistributed beta-actin mRNA to the periphery. gamma-actin mRNA distribution was not altered by serum addition at any time. Both proteins, as identified by immunochemistry with isoform-specific antibodies, were found in similar cellular structures. Serum-stimulated cell motility is mediated through the GTPase signal transduction pathway. We find that an RNA-binding protein, p62, that is part of this pathway, displays a localization pattern similar to beta-actin mRNA. Our results suggest a new biological mechanism which integrates signal transduction with the supply of an architectural component required for membrane remodeling. We propose that active transport of beta-actin mRNA to regions of cell motility is one possible objective of these signal transduction pathways.

3T3 Cells↗
PubMed1994

Regulation of nonmuscle myosin light chain 3 gene expression in response to exogenous MLC3nm mRNA.

We have evaluated the regulation of the nonmuscle alkali myosin light chain isoform, MLC3nm, in mouse C2 myoblasts, in vitro. We altered the normal MLC mRNA profile of these cells, using stable transfection to introduce an exogenous pool of human MLC3nm mRNA. We used an isoform-specific, species-specific mouse MLC3nm cDNA probe to examine the response of the endogenous gene to the exogenous expression. At high cell density, expression of the endogenous mouse MLC3nm mRNA in transfectants is reduced to 50-70% of that in vector-transfected controls. These results suggest that a feedback mechanism operates in vitro, to regulate the size of the total MLC3nm mRNA pool. The down regulation of the mRNA for endogenous isoform is not detected at low cell density, suggesting that the mechanism may be density dependent and related to myoblast differentiation.

Amino Acid Sequence↗
PubMed1994

Induction of anchorage independent growth and serum resistance in immortalized human bronchial epithelial cells by alteration of the cytoskeleton.

Malignant transformation is frequently accompanied by obvious changes in cytoarchitecture, but the importance of these changes has been difficult to assess in view of the large number of other cellular changes that also occur. In this study, we transfected the SV40-immortalized human bronchial epithelial cell line, BEAS-2B, with human wild-type beta or gamma actin gene expression plasmids to induce cytoskeletal changes and to determine whether this was associated with altered cellular growth properties. Cells expressing the exogenous full-length actin genes underwent a fibroblastoid change in morphology which was reflected in changes in their pattern of actin cable organization, and acquired both the ability to grow under anchorage-independent conditions and resistance to the normal growth inhibitory effects of fetal bovine serum. These phenotypic changes correlated with changes in actin mRNA levels, but not with changes in actin protein levels. The phenotypically altered cells were not tumorigenic when injected subcutaneously in athymic nude mice, and they retained the ability to suppress the tumorigenic potential of a lung carcinoma cell line, HuT-292. Therefore, alteration of the cytoskeleton of immortalized human bronchial epithelial cells resulted in the acquisition of some properties commonly found in malignant cells, but did not result in tumorigenicity.

Actins↗
PubMed1993

Nerve-dependent and -independent patterns of mRNA expression in regenerating skeletal muscle.

Muscle regeneration was used to assess the roles of innervation and intrinsic genetic information in the acquisition of predominant slow and fast contractile protein mRNA profiles in adult skeletal muscle. Rat soleus (slow) and EDL (fast) muscles were allowed to regenerate in the presence and absence of their respective slow and fast nerves. Levels of mRNAs encoding fast, slow, and cardiac contractile protein isoforms were quantitatively determined at specific times during regeneration. All muscles initially expressed a heterogeneous pattern of fast, slow, and cardiac isoform mRNAs during myotube formation. Subsequently, the EDL muscle made a transition to a predominantly fast profile of mRNAs which was independent of innervation. The mRNA profile for the noninnervated regenerating soleus muscle was similar to both the innervated and the noninnervated EDL muscle profile. Thus, the decision to express predominantly fast isoform mRNAs is intrinsic to in vivo muscle regeneration with the fast nerve not appearing to be informative. In contrast, acquisition of a slow mRNA profile is dependent on the presence of a slow nerve. The mRNAs encoding slow isoforms from all of the contractile protein gene families are upregulated during the period of reestablishment of neuromuscular transmission. Additionally, there is no concomitant down-regulation of the fast isoform mRNAs upon reinnervation in the soleus regenerate. We propose that information both intrinsic to the muscle and supplied by the in vivo environment acts to provide potential isoform mRNA options to the regenerating muscle.

Animals↗
PubMed1993

Myosin light chain gene expression associated with disease states of the human heart.

During development of the human heart, the atrial isoform of alkali myosin light chain (MLC1A) is expressed in the ventricle. With maturation of the heart, MLC1A expression is completely replaced by that of the adult ventricular myosin light chain, MLC1V. We have evaluated the re-expression of MLC1A as a marker of different disease states of the human ventricle. RNA was isolated from the ventricles of patients with idiopathic dilated cardiomyopathy (CM) and severe congenital cardiac defects (CCD). Northern blot analysis was used to measure the mRNA levels of MLC1A and MLC1V in these samples. As a control, the level of regulatory MLC2V mRNA was also measured. We find that the level of MLC2V mRNA per microgram total ventricular RNA is very similar in CM, CCD and normal human samples. In contrast, we find that MLC1V mRNA levels tend to be reduced in both CM and CCD samples. In the case of the CCD samples, this apparent drop in MLC1V is compensated by expression of the developmental MLC1A isoform. However, in CM patients in end-stage failure, expression of MLC1A is barely detectable. The expression of MLC1A in CCD samples may reflect an adaptive mechanism in response to cardiac overload. The failure to detect substantial MLC1A expression in the CM samples may reflect the failure of such an adaptive mechanism.

Adult↗
PubMed1993

Induction of neuron-specific tropomyosin mRNAs by nerve growth factor is dependent on morphological differentiation.

We have examined the expression of brain-specific tropomyosins during neuronal differentiation. Both TmBr-1 and TmBr-3 were shown to be neuron specific. TmBr-1 and TmBr-3 mRNA levels increased during the most active phase of neurite outgrowth in the developing rat cerebellum. In PC12 cells stimulated by nerve growth factor (NGF) to differentiate to the neuronal phenotype, TmBr-1 and TmBr-3 levels increased with an increasing degree of morphological differentiation. Induction of TmBr-1 and TmBr-3 expression only occurred under conditions where PC12 cells were permitted to extend neurites. NGF was unable to maintain levels of TmBr-1 and TmBr-3 with the loss of neuronal phenotype by resuspension of differentiated PC12 cells. The unique cellular expression and regulation in vivo and in vitro of TmBr-1 and TmBr-3 strongly suggests a critical role of these tropomyosins in neuronal microfilament function. The findings reveal that the induction and maintenance of the neuronal tropomyosins is dependent on morphological differentiation and the maintenance of the neuronal phenotype.

Actin Cytoskeleton↗
PubMed1993

Noncoding regions of the gamma-actin gene influence the impact of the gene on myoblast morphology.

We have addressed the question of whether two highly conserved noncoding regions of the gamma-actin gene are of functional importance. Human gamma-actin gene constructs deleted for either the entire 3' untranslated region (UTR) and 3' flank or intron III sequences were transfected into mouse myoblasts and the resulting clones were analyzed for cell morphology and actin protein expression. Transfectants carrying the gamma-actin gene deleted for the 3' end (gamma 22) exhibited numerous long pseudopods and increased surface area. In contrast, transfectants expressing the gamma-actin gene deleted for intron III (gamma 156) were rounded with blebs over the cell surface and showed decreased surface area. The relative expression of beta- to gamma-actin protein decreased for both transfectant types. The total actin protein levels remained constant for the gamma 22 cells but decreased for the gamma 156 cells. The results indicate that alterations to transfectant cell morphology can be influenced by the presence or absence of different noncoding regions in the transfected gamma-actin gene. The mechanisms by which noncoding regions of the gamma-actin gene influence the impact of the gene are unknown. Nevertheless, these noncoding regions are isoform specific and highly conserved in evolution. We propose that the functional significance of the different actin isoforms may involve the properties of these noncoding regions in addition to the differences in protein sequence.

Actins↗
PubMed1993

Differential regulation of tropomyosin isoform organization and gene expression in response to altered actin gene expression.

Phenotypically altered C2 myoblast cells, generated by the stable transfection of human nonmuscle actin genes (Schevzov, G., C. Lloyd, and P. Gunning. 1992. J. Cell Biol. 117:775-786), exhibit a differential pattern of tropomyosin cellular organization and isoform gene expression. The beta-actin transfectants displaying a threefold increase in the cell surface area, showed no significant changes in the pattern of organization of the high M(r) tropomyosin isoform, Tm 2, or the low M(r) tropomyosin isoform, Tm 5. In contrast, the gamma- and beta sm-actin gene transfectants, exhibiting a twofold decrease in the cell surface area, had an altered organization of Tm 2 but not Tm 5. In these actin transfectants, Tm 2 did not preferentially segregate into stress fiber-like structures and the intensity of staining was greatly diminished. Conversely, a well-defined stress fiber-like organization of Tm 5 was observed. The pattern of organization of these tropomyosin isoforms correlated with their expression such that a profound decrease in Tm 2 expression was observed both at the transcript and protein levels, whereas Tm 5 remained relatively unchanged. These results suggest that relative changes in nonmuscle actin gene expression can affect the organization and expression of tropomyosin in an isoform specific manner. Furthermore, this apparent direct link observed between actin and tropomyosin expression suggests that nonpharmacological signals originating in the cytoskeleton can regulate cytoarchitectural gene expression.

Actins↗
PubMed1993

Beta and gamma actin mRNAs are differentially located within myoblasts.

Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.

Actins↗
PubMed1992

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.

Adult↗
PubMed1992

Molecular cloning and primary structure of the avian Thy-1 glycoprotein.

We have previously characterised, both biochemically and immunohistochemically, a 23 kDa putative avian Thy-1 protein homologue. In this report we have examined the carbohydrates present on the protein and determined the partial protein sequence of enzymatically and CNBr-produced peptides. The protein sequences enabled us to clone an essentially full-length (1854 bp) cDNA using PCR and colony screening of an embryonic day (ED) 18 forebrain pUEX-1 cDNA library. Analysis of deduced amino acid sequence shows the 23 kDa protein to be 110 amino acids in length compared to mouse (112) and human and rat (111) while still retaining the conserved cysteine residues. The N-glycosylation site at position 61 is the same as that in the human protein, but is different from that in the rodent (position 75). Northern blot analysis of Thy-1 mRNA expression in the forebrain, cerebellum and tectum show that all three tissues have low levels at ED4 (forebrain and tectum) and ED8 (cerebellum), rising most rapidly between ED16 and the first few days post-hatch. Analysis of various tissues at hatch and adult show expression to be predominantly neuronal with very low levels in some other organs, mainly at hatch, indicating the possibility of subsets of cells, which we have also seen in histological sections, in these tissues expressing Thy-1 mRNA. Bone marrow and blood cells were also negative for Thy-1 protein.

Aging↗