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Biomedical subjects

Robert D Goldman

Publications and source records attributed to Robert D Goldman.

At least 19 recordsLinked to original sources
PubMed2006

A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death.

The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.

Apoptosis↗
PubMed2006

Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging.

The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a mutant lamin A (LADelta50). Nuclei in cells expressing LADelta50 are abnormally shaped and display a loss of heterochromatin. To determine the mechanisms responsible for the loss of heterochromatin, epigenetic marks regulating either facultative or constitutive heterochromatin were examined. In cells from a female HGPS patient, histone H3 trimethylated on lysine 27 (H3K27me3), a mark for facultative heterochromatin, is lost on the inactive X chromosome (Xi). The methyltransferase responsible for this mark, EZH2, is also down-regulated. These alterations are detectable before the changes in nuclear shape that are considered to be the pathological hallmarks of HGPS cells. The results also show a down-regulation of the pericentric constitutive heterochromatin mark, histone H3 trimethylated on lysine 9, and an altered association of this mark with heterochromatin protein 1alpha (Hp1alpha) and the CREST antigen. This loss of constitutive heterochromatin is accompanied by an up-regulation of pericentric satellite III repeat transcripts. In contrast to these decreases in histone H3 methylation states, there is an increase in the trimethylation of histone H4K20, an epigenetic mark for constitutive heterochromatin. Expression of LADelta50 in normal cells induces changes in histone methylation patterns similar to those seen in HGPS cells. The epigenetic changes described most likely represent molecular mechanisms responsible for the rapid progression of premature aging in HGPS patients.

Aging, Premature↗
PubMed2006

Nuclear lamins, diseases and aging.

Nuclear lamins are type V intermediate filament proteins. They are the major building blocks of the peripheral nuclear lamina, a complex meshwork of proteins underlying the inner nuclear membrane. In addition to providing nuclear shape and mechanical stability, they are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly and nuclear positioning. Over the past few years, interest in the lamins has increased because of the identification of at least 12 distinct human diseases associated with mutations in the LMNA gene, which encodes A-type lamins. These diseases, collectively termed laminopathies, affect muscle, adipose, bone, nerve and skin cells and range from muscular dystrophies to accelerated aging.

Aging↗
PubMed2006

A mitotic lamin B matrix induced by RanGTP required for spindle assembly.

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.

Animals↗
PubMed2006

Tropomyosins are present in lamellipodia of motile cells.

This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Assembling an intermediate filament network by dynamic cotranslation.

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these "translation factories." This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation.

Animals↗
PubMed2005

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

Actin Cytoskeleton↗
PubMed2005

Functions and dysfunctions of the nuclear lamin Ig-fold domain in nuclear assembly, growth, and Emery-Dreifuss muscular dystrophy.

The non-alpha-helical C terminus of Xenopus lamin B3 (LB3T) inhibits the polymerization of lamin B3 in vitro and prevents the assembly of nuclei in Xenopus egg interphase extracts. To more precisely define the functions of LB3T in nuclear assembly, we have expressed subdomains of LB3T and determined their effects on nuclear assembly in Xenopus extracts. The results demonstrate that the Ig-fold motif (LB3T-Ig) is sufficient to inhibit lamin polymerization in vitro. Addition of the LB3T-Ig to egg extracts before the introduction of chromatin prevents chromatin decondensation and the assembly of the lamina, membranes, and pore complexes comprising the nuclear envelope. When added to assembled nuclei, LB3T-Ig prevents the further incorporation of lamin B3 into the endogenous lamina and blocks nuclear growth. The introduction of a point mutation in LB3T-Ig (R454W; LB3T-IgRW), known to cause Emery-Dreifuss muscular dystrophy when present in lamin A, does not inhibit lamin polymerization, chromatin decondensation, or nuclear assembly and growth. These results shed light on the specific alterations in lamin functions attributable to a known muscular dystrophy mutation and provide an experimental framework for revealing the effects of other mutations causing a wide range of laminopathies.

Animals↗
PubMed2005

Intermediate filament proteins participate in signal transduction.

How timely transport of chemical signals between the distal end of long axonal processes and the cell bodies of neurons occurs is an interesting and unresolved issue. Recently, Perlson et al. presented evidence that cleavage products of newly synthesized vimentin, an intermediate filament (IF) protein, interact with mitogen-activated protein (MAP) kinases at sites of axon injury. These IF fragments appear to be required for the transport of these kinases to the cell body along microtubule tracks. The truncated vimentin is instrumental in signal propagation as it provides a scaffold that brings together activated MAP kinases (such as Erk 1 and Erk2), as well as importin beta and cytoplasmic dynein. The authors propose that this all-in-one transport complex has the extraordinary ability to travel towards the cell body and enter the nucleus where the kinases activate and influence gene expression so that a neuron can generate a timely response to injury.

Animals↗
PubMed2005

Keratin 8 phosphorylation by protein kinase C delta regulates shear stress-mediated disassembly of keratin intermediate filaments in alveolar epithelial cells.

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

Adenosine Triphosphate↗
PubMed2005

The nuclear lamina comes of age.

Many nuclear proteins form lamin-dependent complexes, including LEM-domain proteins, nesprins and SUN-domain proteins. These complexes have roles in chromatin organization, gene regulation and signal transduction. Some link the nucleoskeleton to cytoskeletal structures, ensuring that the nucleus and centrosome assume appropriate intracellular positions. These complexes provide new insights into cell architecture, as well as a foundation for the understanding of the molecular mechanisms that underlie the human laminopathies - clinical disorders that range from Emery-Dreifuss muscular dystrophy to the accelerated ageing seen in Hutchinson-Gilford progeria syndrome.

Actins↗
PubMed2005

Nuclear lamins: building blocks of nuclear structure and function.

The cell nucleus is surrounded by a complex membranous envelope which separates the nucleoplasm from the cytoplasm. Unlike the cytoplasm, the nucleoplasm is not subdivided into membrane-bound compartments, which allows for the efficient segregation of a wide range of complex metabolic activities. In the absence of such membrane compartmentalization, the nucleus is faced with the daunting task of efficiently segregating and interconnecting an enormous array of critically important functions. These include the assembly of the large multi-component complexes or 'factories' involved in DNA replication and transcription. These structures are dynamic as they are assembled and disassembled both spatially and temporally at different times, implying the existence of an infrastructure or nucleoskeleton responsible for establishing and maintaining a complex nuclear architecture. There is increasing evidence that the nuclear lamins are essential elements of this nuclear infrastructure, and that their proper assembly and organization are required for numerous essential nuclear functions. Our goal has been to determine the roles of the nuclear lamins in vital nuclear processes including DNA replication and transcription. The hypothesis directing our investigations is that the lamins form a 3D network that courses throughout the nucleoplasm providing an infrastructure for the assembly and distribution of numerous multicomponent complexes involved in a wide range of nuclear functions.

Animals↗
PubMed2004

Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.

Aged↗
PubMed2004

Cdk5 regulates axonal transport and phosphorylation of neurofilaments in cultured neurons.

Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, to influence axonal stability and their maturation to large-caliber axons. Cdk5, a serine/threonine kinase homologous to the mitotic cyclin-dependent kinases, phosphorylates NF subunits in intact cells. In this study, we used two different haptenized NF subunits and manipulated cdk5 activity by microinjection, transfection and pharmacological inhibition to monitor the effect of Cdk5-p35 on NF dynamics and transport. We demonstrate that overexpression of cdk5 increases NF phosphorylation and inhibits NF axonal transport, whereas inhibition both reduces NF phosphorylation and enhances NF axonal transport in cultured chicken dorsal-root-ganglion neurons. Large phosphorylated-NF 'bundles' were prominent in perikarya following cdk5 overexpression. These findings suggest that Cdk5-p35 activity regulates normal NF distribution and that overexpression of Cdk5-p35 induces perikaryal accumulation of phosphorylated-NFs similar to those observed under pathological conditions.

Animals↗
PubMed2004

Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments.

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the (32)P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

Amino Acid Sequence↗
PubMed2004

Intermediate filaments are dynamic and motile elements of cellular architecture.

Recent evidence showing that intermediate filaments (IFs) are dynamic, motile elements of the cytoskeletal repertoire of vertebrate cells has overturned the long-standing view that they simply form static 'space filling' cytoplasmic networks. In fact, many types of IF are now known to engage in a remarkable array of movements that are closely associated with their assembly, disassembly and subcellular organization. Some of these motile properties are intrinsic to IFs and others are attributable to molecular crosstalk with either microtubules or actin-containing microfilaments. This crosstalk is, to a large extent, mediated by molecular motors, including conventional kinesin and cytoplasmic dynein. These motors are responsible for the high-speed delivery of nonfilamentous IF precursors and short filaments to specific regions of the cytoplasm, where they assemble into long IFs. Interestingly, the patterns and speeds of IF movements vary in different cell types and even within different regions of the same cell. These differences in motility may be related to their interactions with different types of molecular motor and/or other factors, such as IF-associated proteins.

Actin Cytoskeleton↗
PubMed2004

Intermediate filaments mediate cytoskeletal crosstalk.

Intermediate filaments, actin-containing microfilaments and microtubules are the three main cytoskeletal systems of vertebrate and many invertebrate cells. Although these systems are composed of distinctly different proteins, they are in constant and intimate communication with one another. Understanding the molecular basis of this cytoskeletal crosstalk is essential for determining the mechanisms that underlie many cell-biological phenomena. Recent studies have revealed that intermediate filaments and their associated proteins are important components in mediating this crosstalk.

Actin Cytoskeleton↗