Perspectives on herpes-APP interactions.
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Alzheimer Disease↗
Biomedical subjects
E L Bearer
Publications and source records attributed to E L Bearer.
Actin dynamics in platelets.
The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps which is reproducible in vitro. The actin dynamics underlying these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-Actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.
Actins↗
The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines.
Recent advances in microbiology implicate the cytoskeleton in the life cycle of some pathogens, such as intracellular bacteria, Rickettsia and viruses. The cellular cytoskeleton provides the basis for intracellular movements such as those that transport the pathogen to and from the cell surface to the nuclear region, or those that produce cortical protrusions that project the pathogen outwards from the cell surface towards an adjacent cell. Transport in both directions within the neuron is required for pathogens such as the herpesviruses to travel to and from the nucleus and perinuclear region where replication takes place. This trafficking is likely to depend on cellular motors moving on a combination of microtubule and actin filament tracks. Recently, Bearer et al. reconstituted retrograde transport of herpes simplex virus (HSV) in the giant axon of the squid. These studies identified the tegument proteins as the viral proteins most likely to recruit retrograde motors for the transport of HSV to the neuronal nucleus. Similar microtubule-based intracellular movements are part of the biological behavior of vaccinia, a poxvirus, and of adenovirus. Pathogen-induced surface projections and motility within the cortical cytoplasm also play a role in the life cycle of intracellular pathogens. Such motility is driven by pathogen-mediated actin polymerization. Virulence depends on this actin-based motility, since virulence is reduced in Listeria ActA mutants that lack the ability to recruit Arp2/3 and polymerize actin and in vaccinia virus mutants that cannot stimulate actin polymerization. Inhibition of intracellular movements provides a potential strategy to limit pathogenicity. The host cell motors and tracks, as well as the pathogen factors that interact with them, are potential targets for novel antimicrobial therapy.
Actins↗
Motility-related proteins as markers for head and neck squamous cell cancer.
HYPOTHESIS: Increased cell motility is a hallmark of cancer cells. Proteins involved in cell motility may be used as molecular markers to characterize the malignant potential of tumors. METHODS: Molecular biology and immunohistochemistry techniques were used to investigate the expression of a selected panel of motility-related proteins (Rho A, Rac 2, Cdc42, PI3K, 2E4, and Arp2) in normal, premalignant, and squamous cell cancer cell lines of human head and neck origin. To assess the clinical potential of these proteins as molecular markers for cancer, immunohistochemistry was performed on paraffin-fixed head and neck cancer specimens (n = 15). RESULTS: All six motility-associated proteins were overexpressed in the premalignant and squamous cell cancer cell lines relative to normal keratinocytes. Immunohistochemistry with Rho A and Rac 2 showed increased staining in areas of cancer but not in normal tissue. CONCLUSION: Proteins involved in cell motility can be used as markers for head and neck squamous cell carcinoma. The head and neck cell lines used in this study may be used as a model to further investigate cell motility. Molecular markers of motility could have a significant impact on the diagnosis and staging of cancers originating from differentiated non-motile cells.
Biomarkers, Tumor↗
Microsequencing of myosins for PCR primer design.
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Animals↗
Retrograde axonal transport of herpes simplex virus: evidence for a single mechanism and a role for tegument.
Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid, Loligo pealei, a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 +/- 0.26 micrometer/sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that (i) HSV recruits the squid retrograde transport machinery; (ii) viral tegument and capsid but not envelope are sufficient for this recruitment; and (iii) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit.
Animals↗
VASP protects actin filaments from gelsolin: an in vitro study with implications for platelet actin reorganizations.
An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament-severing protein in platelets. Shape change is inhibited by VASP, another abundant actin-binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASP's known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolin's ability to disassemble actin filaments in a dose-dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP-actin filaments at least as well as to actin alone. VASP inhibited gelsolin-induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASP's affinity for actin (K(d) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton.
Actins↗
2E4/Kaptin (KPTN)--a candidate gene for the hearing loss locus, DFNA4.
Stereocilia of the inner ear play an integral role in the mechanotransduction of sound. Their structural support is derived from actin filaments and actin-binding proteins. We have identified a novel actin-binding protein, 2E4-kaptin (KPTN), which appears to be involved in this structural network. Using double label immunofluorescence, we now show that KPTN extends beyond the barbed ends of actin filaments at the tips of stereocilia, and using cloned human cDNA, we mapped KPTN to chromosome 19q13.4. A combination of FISH, radiation hybrid mapping and YAC screening localized KPTN between markers D19S412 and NIB1805, making this gene an excellent functional and positional candidate for DFNA4, a form of autosomal dominant non-syndromic hearing loss. We identified a second family with inherited deafness that also maps to the DFNA4 region. To screen KPTN for deafness-causing mutations, we first determined its genomic structure and then completed a mutational analysis by direct sequencing and SSCP in affected family members. Although no deafness-causing mutations were identified in the coding region, KPTN remains an excellent candidate gene for hearing loss; by synteny, its murine orthologue also remains a candidate gene for the Nijmegan waltzer (nv) mouse mutant, which has vestibular defects and a variable sensorineural hearing loss.
Adult↗
2E4 (kaptin): a novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear.
Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein, 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner ear to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation.
Actins↗
Association of actin filaments with axonal microtubule tracts.
Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.
Actin Cytoskeleton↗
Squid axoplasm supports the retrograde axonal transport of herpes simplex virus.
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Animals↗
Primary peptide sequences from squid muscle and optic lobe myosin IIs: a strategy to identify an organelle myosin.
The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60-95%) and chick pectoralis muscle (31-83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50-60%, identities), and some also matched unconventional myosins (33-50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.
Amino Acid Sequence↗
An axoplasmic myosin with a calmodulin-like light chain.
Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
Amino Acid Sequence↗
Actin-based motility of isolated axoplasmic organelles.
We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.
Actins↗
Cytoskeletal domains in the activated platelet.
Platelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin-binding proteins in platelets, filamentous (F)-actin affinity chromatography was used. This approach identified > 30 different proteins from ADP-activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin-binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninety-three percent of these proteins (13 of 14 proteins tested) were found to be associated with actin-rich structures in vivo. Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin-binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin-based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin-based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process.
3T3 Cells↗
Changes in tyrosine-phosphorylated p190 and its association with p120 type I and p100 type II rasGAPs during myelomonocytic differentiation of human leukemic cells.
A M(r) 190,000 protein (p190) functions as a GTPase-activating protein (GAP) for Rho and Rac family proteins, which are involved in regulating cytoskeletal actin and membrane ruffling. Tyrosine-phosphorylated p190 also complexes with rasGAP, a regulator of Ras activity, thus possibly linking Ras and Rho pathways. Leukemic cells induced to differentiate along myelomonocytic lineages have increased filamentous actin (as evidenced by phalloidin staining) and extended pseudopodia, and become irregularly shaped and flattened, suggesting altered Rho and Rac function. We, therefore, hypothesized that changes in p190 and its association with rasGAP are an integral part of these shape changes. During phorbol 13-myristate 25-acetate-induced monocytic differentiation of HL60 promyelocytic and RWLeu4 chronic myelogenous leukemic cells, the total amount of p190 decreases rapidly but returns to initial levels by 12 h. In RWLeu4, this was accompanied by commensurate changes in p190 tyrosine phosphorylation and association with p120 type I rasGAP. Association of p190 and type I rasGAP was demonstrated by immunoprecipitation with antibodies to either protein. An additional band at M(r) 100,000 (p100) was detected in immunoprecipitates after 12 h of phorbol 13-myristate 25-acetate treatment. Reverse transcription-PCR and immunoblot analyses suggest that p100 is type II rasGAP, an alternatively spliced product of p120 type I rasGAP. p100 was expressed only in response to direct protein kinase C activators, but all classes of differentiation agents increased tyrosine-phosphorylated p190. Rho and Rac are known to be involved in regulating actin polymerization. The results presented here show that the association of p190 with type I rasGAP parallels increases in actin polymerization and cell adhesion. This suggests a role for p190-rasGAP interactions in phorbol 13-myristate 25-acetate-induced cytoskeletal reorganization.
Base Sequence↗
Distribution of Xrel in the early Xenopus embryo: a cytoplasmic and nuclear gradient.
In Drosophila embryos, the dorsal gene, a member of the rel family of transcription factors, is a maternal gene required for dorsal/ventral axis specification and mesoderm and neural migration and differentiation. In this report, the presence and distribution of Xrel-1 protein, a Xenopus homologue of the rel family of transcription factors, is described during early embryogenesis. Antiserum to v-rel, an avian homologue of dorsal, was used in immunoblot to detect rel proteins in Xenopus embryos. Anti-v-rel serum recognized a single band of approximately 57 kDa in Western blots of extracts of Xenopus embryos. Antiserum was also generated against bacterially expressed Xrel-1 fusion protein, a Xenopus rel homologue expressed during oogenesis. Anti-Xrel-1 antiserum recognized the same approximately 57 kDa band in Western blots as anti-v-rel. In developmental Westerns, this 57 kDa protein was present throughout early embryogenesis. Whole mounts and histologic sections of staged embryos were stained with anti-rel antiserum. Asymmetric staining of the cytoplasm of the animal cap became apparent by the eight-cell stage, with little or no staining of the vegetal hemisphere. Staining of nuclei in cells of the animal cap down to the equatorial zone became apparent between Stage 7 and 8, while vegetal nuclei never stained. Unlike Drosophila dorsal, no dorsal-ventral gradient of Xrel-1 could be detected. Anti-actin antibodies stained embryos uniformly, while preimmune serum or anti-v-rel antiserum immunoabsorbed against bacterially expressed Xrel-1 protein failed to stain. Nuclear staining diminished during gastrulation, but reappeared during neurulation. In conclusion, Xrel-1 protein is enriched in the cytoplasm of the animal pole of early Xenopus embryos, and enters the nuclei of the cells of the animal cap and presumptive mesoderm at Stage 7-1/2. The presence of this putative transcription factor in the nuclei of these cells prior to mid-blastula transition suggests that Xrel-1 may be involved in programming animal cells to respond to vegetal-inducing factors.
Animals↗
Evidence for myosin motors on organelles in squid axoplasm.
Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.
Acrosome↗