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R D Goldman

Publications and source records attributed to R D Goldman.

At least 73 records · Page 4Linked to original sources
PubMed1995

The dynamic properties and possible functions of nuclear lamins.

The nuclear lamins are thought to form a thin fibrous layer called the nuclear lamina, underlying the inner nuclear envelope membrane. In this review, we summarize data on the dynamic properties of nuclear lamins during the cell cycle and during development. We discuss the implications of dynamics for lamin functions. The lamins may be involved in DNA replication, chromatin organization, differentiation, nuclear structural support, and nuclear envelope reassembly. Emphasis is placed on recent data that indicate that the lamina, contrary to previous views, is not a static structure. For example, the lamins form nucleoplasmic foci, distinct from the peripheral lamina, which vary in their patterns of distribution as well as their composition in a cell cycle-dependent manner. During the S phase, these foci colocalize with chromatin and sites of DNA replication. At other points during the cell cycle, they may represent sites of lamin post-translation processing that take place prior to incorporation into the lamina. Secondary modifications of the lamins such as isoprenylation and phosphorylation are involved in the regulation of the dynamic properties and the assembly of lamins. In addition, a number of lamin-associated proteins have been recently identified and these are described along with their potential functions.

Amino Acid Sequence↗
PubMed1995

Quantitative comparison of two distinct echogenic structures appearing on the same image using gain-assisted densitometric evaluation of sonograms (GADES).

Gain-assisted densitometric evaluation of sonograms with two distinct echogenic structures appearing on each image was performed to establish if their density-gain setting curves have similar inclinations. We used the breast model (n = 67) in which hyperechoic masses were compared to the surrounding hyperechoic tissue at the optimal gain (Gopt) and at Gopt + 5. Both structures had significantly different film-corrected densities and showed a linear decline of density as the system gain increased from Gopt - 5 to Gopt + 5. The least squares regression lines were almost parallel. The density-gain unit decrease from Gopt - 5 to Gopt was not different, but the decrease from Gopt to Gopt + 5 reached statistical significance. The data suggest that skewed values are theoretically possible in comparing two distinct echogenic structures. However, within the gain intervals used in GADES, these differences are negligible and comparisons are as accurate as with the assessment of a single echogenic structure.

Breast Neoplasms↗
PubMed1995

Echogenicity of fibroadenoma and carcinoma of the breast. Quantitative comparison using gain-assisted densitometric evaluation of sonograms.

The purpose of this study was to evaluate the role of ultrasonography in differentiation between the echogenic characteristics of carcinoma and those of fibroadenoma of the breast. Two mathematical transformations of the measured density on a sonographic image of the breast lesion at three different system gain settings were used in cases of carcinoma (n = 16) and fibroadenoma (n = 31) to provide standardized density values. A significant correlation was found between the tumor and the surrounding tissue densities in both carcinomas (r = 0.77, P = 0.0004) and fibroadenomas (r = 0.79, P < 0.000001. The mean standardized density of the tumor was significantly higher (P < 0.0001) than the surrounding tissue in both carcinomas (2.03 +/- 0.64 D versus 1.23 +/- 0.38 D) and fibroadenomas (2.09 +/- 0.48 D versus 1.33 +/- 0.3 D). However, the differences between density values of carcinoma and fibroadenomas were not significantly different. The data suggest that it may not be possible to differentiate breast carcinoma and fibroadenomas on the basis of the echogenicity of solid breast masses.

Adolescent↗
PubMed1994

IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions.

The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.

Amino Acid Sequence↗
PubMed1994

Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication.

The nuclear lamins form a fibrous structure, the nuclear lamina, at the periphery of the nucleus. Recent results suggest that lamins are also present as foci or spots in the nucleoplasm at various times during interphase of the cell cycle (Goldman, A. E., R. D. Moir, M. Montag-Lowy, M. Stewart, and R. D. Goldman. 1992. J. Cell Biol. 104:725-732; Bridger, J. M., I. R. Kill, M. O'Farrell, and C. J. Hutchison. 1993. J. Cell Sci. 104:297-306). In this report we demonstrate that during mid-late S-phase, nuclear foci detected with lamin B antibodies are coincident with sites of DNA replication as detected by the colocalization of sites of incorporation of bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA). The relationship between lamin B and BrDU is not maintained in the following G1 stage of the cell cycle. Furthermore, the nuclear staining patterns seen with antibodies directed against lamins A and C in mid-late S-phase do not coalign with the lamin B/BrDU-containing structures. These results imply that there is a role for lamin B in the organization of replicating chromatin during S phase.

3T3 Cells↗
PubMed1993

Lamin dynamics.

Nuclear lamins form a highly insoluble structure, the nuclear lamina, which is associated with the nuclear envelope. Recent results suggest, however, that the lamins are more dynamic than originally thought. They accumulate in nucleoplasmic foci in the G1 stage of the cell cycle and later appear mainly in the peripheral lamina. Some of the lamin foci are closely associated with heterochromatin. Furthermore, the various lamin types assemble into the lamina polymer independently of each other. Both the assembly and disassembly of the lamins, as well as the interaction of the lamins with other nuclear structures such as the nuclear membrane, may be mediated by phosphorylation and dephosphorylation.

Animals↗
PubMed1993

Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo.

Keratin intermediate filaments (IF) are obligate heteropolymers containing equal amounts of type I and type II keratin. We have previously shown that microinjected biotinylated type I keratin is rapidly incorporated into endogenous bundles of keratin IF (tonofilaments) of PtK2 cells. In this study we show that the earliest steps in the assembly of keratin subunits into tonofilaments involve the extremely rapid formation of discrete aggregates of microinjected keratin. These are seen as fluorescent spots containing both type I and type II keratins within 1 min post-injection as determined by double label immunofluorescence. These observations suggest that endogenous type II keratin subunits can be rapidly mobilized from their endogenous state to form complexes with the injected type I protein. Furthermore, confocal microscopy and immunogold electron microscopy suggest that the type I-type II keratin spots from in close association with the endogenous keratin IF network. When the biotinylated protein is injected at concentrations of 0.3-0.5 mg/ml, the organization of the endogenous network of tonofilaments remains undisturbed during incorporation into tonofilaments. However, microinjection of 1.5-2.0 mg/ml of biotinylated type I results in significant alterations in the organization and assembly state of the endogenous keratin IF network soon after microinjection. The results of this study are consistent with the existence of a state of equilibrium between keratin subunits and polymerized keratin IF in epithelial cells, and provide further proof that IF are dynamic elements of the cytoskeleton of mammalian cells.

Animals↗
PubMed1992

Cell cycle-dependent changes in the organization of an intermediate filament-associated protein: correlation with phosphorylation by p34cdc2.

During mitosis in BHK-21 baby hamster kidney cells the hyperphosphorylation of the type III intermediate filament (IF) protein vimentin is accompanied by the disruption of the IF network into punctate, protofilamentous structures. In this study, the morphological and biochemical changes of IFAP 300, a 300-kDa IF-crossbridging protein, are examined during mitosis. Double-label immunofluorescence shows that the distribution of IFAP 300 coincides with the typical filamentous pattern displayed by vimentin in interphase cells, whereas in mitotic cells it is reorganized into a punctate, nonfilamentous pattern. Accompanying these latter morphological changes, IFAP 300 is phosphorylated at a unique, mitosis-specific site. Comparison of the sites phosphorylated in cultured cells with those phosphorylated in vitro by various kinases suggests that IFAP 300 is phosphorylated by the same two kinases that phosphorylate vimentin during mitosis. One of these is p34cdc2 protein kinase, which appears to be responsible for the phosphorylation of the mitosis-specific site. The other kinase phosphorylates IFAP 300 in vitro at a site that is also found in the protein immunoprecipitated from either mitotic or interphase cells. In contrast to vimentin, the phosphorylation levels of IFAP 300 are not obviously altered between interphase and mitosis. Our results show that IFAP 300 is a physiological substrate for p34cdc2 and that this kinase may be involved in the mitotic reorganization of IFAP 300 by phosphorylating a mitosis-specific site. Taken together with our previous results, this study suggests that the activation of p34cdc2 coordinates the mitotic reorganization of the vimentin IF network both by severing IF-IF connections mediated by IFAP 300 and by disassembling individual IFs into protofilaments.

Animals↗
PubMed1992

Cytoskeletal integrity in interphase cells requires protein phosphatase activity.

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

Animals↗
PubMed1992

Distinct developmental subtypes of cultured non-stellate rat astrocytes distinguished by a new glial intermediate filament-associated protein.

The nature and tissue origin of cultured non-stellate astrocytes have not been defined. On the basis of immunofluorescence microscopy using multiple double-labeling with antibodies to glial fibrillary acidic protein (GFAP), vimentin, and the recently identified intermediate filament-associated protein (IFAP)-70/280kD, four distinct astrocytic subtypes were definable in neonatal rat brain astrocytes in culture. All of these were of the non-stellate type on the basis of morphology. Similar examination of developing rat cerebral cortex identified these same subtypes as distinct differentiation states of astrocytes. These findings indicate that the parallel developmental events can be studied in vitro.

Animals↗
PubMed1992

Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD.

A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.

Animals↗
PubMed1992

Intermediate filament dynamics.

The view of intermediate filaments as static cytoskeletal elements is changing. Studies of exogenous intermediate filament proteins, either microinjected or expressed from transfected genes, have demonstrated that a continuous incorporation of subunits into the polymerized filaments is taking place. This incorporation appears to be required for maintaining normal cytoplasmic networks of intermediate filaments. At the post-translational level, phosphorylation is an important factor in regulating dynamic aspects of intermediate filament organization and structure.

Animals↗
PubMed1992

Intermediate filaments: not so tough after all.

The dynamic properties of cellular protein polymers such as microtubules and microfilaments depend to a large extent on the cell's capacity to modify rapidly the exchange rate between polymerized and unpolymerized pools of subunits. Until quite recently the dynamic nature of intermediate filaments was underestimated because of their biochemical stability in vitro and a paucity of studies on their characteristics in vivo. However, the recent studies described in this review show that the karyoskeletal and cytoskeletal structures that assemble from many intermediate filament proteins possess the properties expected of dynamic protein polymer networks.

Journal Article↗
PubMed1992

Steady state dynamics of intermediate filament networks.

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.

3T3 Cells↗
PubMed1992

Pathway of incorporation of microinjected lamin A into the nuclear envelope.

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.

3T3 Cells↗
PubMed1991

Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid.

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.

Blotting, Western↗
PubMed1991

Some neural intermediate filaments contain both peripherin and the neurofilament proteins.

Mammalian neurons and neuron-like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)-L, NF-M, NF-H, and peripherin. To determine whether these proteins are found within the same 10-nm filament, light and electron microscope immunocytochemistry using peripherin and NF-specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double-label immunofluorescence showed that peripherin and NF-L, -M, and -H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF-containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF-L or peripherin/NF-M/-H double-label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.

Amino Acid Sequence↗