Biomedical subjects

R D Goldman

Publications and source records attributed to R D Goldman.

At least 19 recordsLinked to original sources

PubMed1997

Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

Animals↗

PubMed1997

Laminin-5 and modulation of keratin cytoskeleton arrangement in FG pancreatic carcinoma cells: involvement of IFAP300 and evidence that laminin-5/cell interactions correlate with a dephosphorylation of alpha 6A integrin.

Under normal culture conditions, epithelial cells of the FG line, derived from a pancreatic tumor, characteristically grow in mounds and fail to flatten efficiently onto their substrate. In such cells, keratin intermediate filaments (IFs) are concentrated in the perinuclear region. Furthermore, the IF associated protein, IFAP300, primarily localizes along these keratin bundles. Additionally, alpha 6 beta 4 integrin heterodimers localize in streaks or spots towards the edges of cells while alpha 3 beta 1 integrin is predominantly at cell-cell surfaces. Neither show any obvious interaction with IF. Remarkably, upon plating FG cells into medium containing soluble rat laminin-5, FG cells rapidly adhere and spread onto their substrate. Moreover, FG cells "capture" rat laminin-5 and place it basally in circles or arcs at areas of cell-substrate interaction. Double label immunofluorescence microscopy reveals colocalization of IFAP300 as well as alpha 6 beta 4 and alpha 3 beta 1 integrin with the polarized laminin-5. Concomitantly, alpha 6 integrin undergoes dephosphorylation on serine residue 1041. Laminin-5-induced rapid adhesion can be blocked by antibodies against the alpha 3 integrin subunit. In contrast, while alpha 6 integrin antibodies do not block laminin-5-induced rapid adhesion, they prevent FG cells from assuming an epithelial-like morphology. Keratin IF bundles associate with IFAP300-alpha 6 beta 4/alpha 3 beta 1 integrin complexes along the cell-substratum-attached surface of FG cells coincubated in laminin-5-containing medium. Coprecipitation results suggest that in these complexes, IFAP300 may associate with the alpha 6 beta 4 integrin heterodimer. Based on our results and published evidence that IFAP300 binds keratin in vitro [Skalli et al., 1994; J. Cell Biol. 125:159-170], we propose that laminin-5/FG cell interaction results in a novel integrin dephosphorylation event, which subsequently induces IFAP300 association with alpha 6 beta 4 integrin. IFAP300 then mediates the interaction of IFs with the cell surface via the alpha 6 beta 4 integrin heterodimer.

Amino Acid Sequence↗

PubMed1997

Intermediate filaments and cytoplasmic networking: new connections and more functions.

Recent research highlights the roles of cytoskeletal intermediate filaments (IFs) and their interactions with both the cell surface and other cytoskeletal systems in maintaining cellular integrity and the mechanical properties of cytoplasm. This has been demonstrated by analyses of mutations in IF-associated proteins (IFAPs) that are involved in connecting IFs to cell surface junctions. New data also point to the role of IFAPs as molecular 'nuts and bolts' in the construction of an integrated cytoplasmic architecture. This is highlighted by the initial descriptions of a family of multifunctional molecules that are capable of bridging IFs to other cytoskeletal elements. These findings, together with the development of specific peptide inhibitors capable of disassembling IF networks in vivo, are paving the way to the identification of new cellular functions for IFs and IFAPs.

Cytoplasm↗

PubMed1997

Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments.

The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of microcystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmoplakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed morphological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmodulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.

Actin Cytoskeleton↗

PubMed1996

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Amino Acid Sequence↗

PubMed1996

The function of intermediate filaments in cell shape and cytoskeletal integrity.

This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.

3T3 Cells↗

PubMed1996

The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells.

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

Animals↗

PubMed1996

Alterations in neural intermediate filament organization: functional implications and the induction of pathological changes related to motor neuron disease.

The properties regulating the supramolecular organization of neural intermediate filament (NIF) networks have been investigated in cultured dorsal root ganglion (DRG) neurons. The studies described take advantage of the ability of endogenous NIF to incorporate purified biotinylated neurofilament triplet (NFT) proteins, NF-L, NF-M and NF-H. When injected at concentrations of 0.8-1.0 mg/ml injection buffer, each of these proteins is incorporated without perturbing the endogenous NIF network. However, at progressively higher concentrations, NF-H induces the aggregation and accumulation of NIF in the cell body. Subsequent to the induction of these aggregates, numerous alterations in the cytoarchitecture of neurons can be detected. The latter occur in a temporal sequence which appears to begin with the fragmentation of the Golgi complex. At later times, accumulation of mitochondria within the proximal region of neurites, peripheralization of the nucleus, and a significant decrease in neurite caliber become obvious. After longer time periods, the NIF aggregates are seen to react with an antibody which reveals abnormally phosphorylated NF-H. These observations demonstrate that an imbalance in the normal stoichiometric relationships among the NFT proteins rapidly alters the supramolecular organization of the NIF network. These changes most likely reflect the normal functions of neurofilaments in cell shape and the organization and cytoplasmic distribution of membranous organelles. Interestingly, virtually all of these changes closely resemble those which have been reported in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). These findings suggest that cultured neurons can be used as models for more precisely defining the relationships between the formation of NIF aggregates and the sequence of cytopathological events which typify neurodegenerative diseases.

Amyotrophic Lateral Sclerosis↗

PubMed1995

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.

Amino Acid Sequence↗

PubMed1995

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Animals↗

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↗