Colocalization Of Actin Research Articles

Increased expression of secretory actin-binding protein on human spermatozoa is associated with poor semen quality

Antibodies to human sperm are useful diagnostic reagents for detection of changes in sperm protein expression and their relationship with sperm defects and male infertility. The specificity of Hs-16 monoclonal antibody (mAb) and the localization and frequency of the occurrence of Hs-16-recognized protein on human spermatozoa were investigated. Samples from 30 fertile men with normal spermiograms and 30 men with pathological spermiograms were studied. The specificity of Hs-16 mAb was analysed by the western blotting technique and matrix-assisted laser desorption/ionization mass spectrometry. Indirect immunofluorescence with Hs-16 antibody was used to test sperm ejaculates. The Hs-16 antibody detected a human sperm and seminal plasma protein, which was determined to be secretory actin-binding protein (SABP). This specificity was also verified by co-localization of SABP and actin on spermatozoa with Hs-16 and anti-actin antibodies, and partial co-localization of these proteins was found. SABP was localized on the sperm tail, mainly in the midpiece of the tail. Other parts of spermatozoa were labelled with lower frequency. A significant difference was found in SABP labelling between men with normal spermiograms and donors with asthenozoospermia or oligoasthenoteratozoospermia (both P < 0.01), and asthenozoospermia versus oligoasthenoteratozoospermia (P < 0.05). Increased expression of SABP was observed in men with pathological spermiograms. Hs-16 antibody reacts specifically with SABP. SABP can serve as a marker of defective sperm and may be associated with fertility failure.

Migration of bone marrow stromal cells in 3D: 4 Color methodology reveals spatially and temporally coordinated events

The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.

Dual effects of staurosporine on A431 and NRK cells: Microfilament disassembly and uncoordinated lamellipodial activity followed by cell death

The general protein kinase inhibitor staurosporine (STS) has dual effects on human epidermoid cancer cells (A431) and normal rat kidney fibroblasts (NRK). It almost immediately stimulated increased lamellipodial activity of both cell lines and after 2 h induced typical signs of apoptosis, including cytoplasmic condensation, nuclear fragmentation, caspase-3 activation and DNA degradation. In the early phase we observed disruption of actin-containing stress fibres and accumulation of monomeric actin in the perinuclear region and cell nucleus. Increased lamellipodial-like extensions were observed particularly in A431 cells as demonstrated by co-localisation of actin and Arp2/3 complex, whereas NRK cells shrunk and exhibited numerous thin long extensions. These extensions exhibited uncoordinated centrifugal motile activity that appeared to tear the cells apart. Both cofilin and ADF were translocated from perinuclear regions to the cell cortex and, as expected in the presence of a kinase inhibitor, all the cofilin was dephosphorylated. Myosin II was absent from the extensions, and a reduction of phosphorylated myosin light chains was observed within the cytoplasm indicating myosin inactivation. Microtubules and intermediate filaments retained their characteristic filamentous organisation after STS exposure even when the cells became rounded and disorganised. Simultaneous treatment of NRK cells with STS and the caspase inhibitor zVAD did not inhibit the morphological and cytoskeletal changes. However, the cells underwent cell death as verified by positive annexin-V-staining. Thus it seems likely that cell death induced by STS may not only be a consequence of the activation of caspase, instead the disruption of the many motile processes involving the actin cytoskeleton may by itself suffice to induce caspase-independent cell death.

Synthetic D-amino acid peptide inhibits tumor cell motility on laminin-5

Cell motility is partially dependent on interactions between the integrins and the extracellular matrix. Our previous studies have identified synthetic D-amino acid cell adhesion peptides using a combinatorial screening approach. In this study, we demonstrate that HYD1 (kikmviswkg) completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with alpha6beta1 (not alpha6beta4) and alpha3beta1 integrins and surprisingly elevates laminin-5-dependent intracellular signals including focal adhesion kinase, mitogen-activated protein kinase kinase and extracellular signal-regulated kinase. HYD1 does not contain a previously characterized binding sequence for integrins. A scrambled derivative of HYD1, called HYDS (wiksmkivkg), does not interact with the alpha6 or alpha3 integrin subunits and is not biologically active. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal-dependent migration.

The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Mechanisms of xenon‐ and isoflurane‐induced preconditioning – a potential link to the cytoskeleton via the MAPKAPK‐2/HSP27 pathway

We previously demonstrated that the anesthetic gas xenon exerts cardioprotection by preconditioning in vivo via activation of protein kinase C (PKC)-epsilon and p38 mitogen-activated protein kinase (MAPK). P38 MAPK interacts with the actin cytoskeleton via the MAPK-activated protein kinase-2 (MAPKAPK-2) and heat-shock protein 27 (HSP27). The present study further elucidated the underlying molecular mechanism of xenon-induced preconditioning (Xe-PC) by focusing on a potential link of xenon to the cytoskeleton. Anesthetized rats received either xenon (Xe-PC, n = 6) or the volatile anesthetic isoflurane (Iso-PC, n = 6) during three 5-min periods interspersed with two 5-min and one final 10-min washout period. Control rats (n = 6) remained untreated for 45 min. Additional rats were either pretreated with the PKC inhibitor Calphostin C (0.1 mg kg(-1)) or with the p38 MAPK inhibitor SB203580 (1 mg kg(-1)) with and without anesthetic preconditioning (each, n = 6). Hearts were excised for immunohistochemistry of F-actin fibers and phosphorylated HSP27. Phosphorylation of MAPKAPK-2 and HSP27 were assessed by Western blot. HSP27 and actin colocalization were investigated by co-immunoprecipitation. Xe-PC induced phosphorylation of MAPKAPK-2 (control 1.0 +/- 0.2 vs Xe-PC 1.6 +/- 0.1, P < 0.05) and HSP27 (control 5.0 +/- 0.5 vs Xe-PC 9.8 +/- 1.0, P < 0.001). Both effects were blocked by Calphostin C and SB203580. Xe-PC enhanced translocation of HSP27 to the particulate fraction and increased F-actin polymerization. F-actin and pHSP27 were colocalized after Xe-PC. Xe-PC activates MAPKAPK-2 and HSP27 downstream of PKC and p38 MAPK. These data link Xe-PC to the cytoskeleton, revealing new insights into the mechanisms of Xe-PC in vivo.

The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K+ current

Multiple cell-signaling pathways converge to modulate large-conductance, voltage- and Ca2+-sensitive K+ channel (maxi-K channel) activity and buffer cell excitability in human myometrial smooth muscle cells (hMSMCs). Recent evidence indicates that maxi-K channel proteins can target to membrane microdomains; however, their association with other proteins within these macromolecular complexes has not been elucidated. Biochemical isolation of detergent-resistant membrane fractions from human myometrium demonstrates the presence of maxi-K channels in lipid raft microdomains, which cofractionate with caveolins. In both nonpregnant and late-pregnant myometrium, maxi-K channels associate and colocalize with caveolar scaffolding proteins caveolin-1 and caveolin-2, but not caveolin-3. Disruption of cultured hMSMC caveolar complexes by cholesterol depletion with cyclodextrin increases an iberiotoxin-sensitive K+ current. Co-immunoprecipitations have indicated that the maxi-K channel also is associated with both alpha- and gamma-actin. Immunocytochemical analysis indicates colocalization of maxi-K channels, actin, and caveolin-1 in primary cultures of hMSMCs. Further experiments using immunoelectron microscopy have shown the proximity of both actin and the maxi-K channel within the same cell surface caveolar structures. Functionally, disruption of the actin cytoskeleton in cultured hMSMCs by cytochalasin D and latrunculin A greatly increased the open-state probability of the channel, while stabilization of actin cytoskeleton with jasplakinolide abolished the effect of latrunculin A. These data indicate that the actin cytoskeleton is involved as part of a caveolar complex in the regulation of myometrial maxi-K channel function.

Host cell tropism underlies species restriction of human and bovine Cryptosporidium parvum genotypes.

It has been recognized recently that human cryptosporidiosis is usually caused by Cryptosporidium parvum genotype I ("human" C. parvum), which is not found in animals. Compared to C. parvum genotype II, little is known of the biology of invasion of the human-restricted C. parvum genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among C. parvum genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of C. parvum genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem. C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes. C. parvum genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast, C. parvum genotype I invaded only human small-bowel cells. The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C parvum genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.

Actin is closely associated with RNA polymerase II and involved in activation of gene transcription

Biochemical and morphological studies have demonstrated the presence of actin in the nucleus of different eukaryotic cells, whereas its role remains unclear. In this work, we studied the interaction and the functional relationship between nuclear actin and RNA polymerase II (RNAP II). The immunofluorescence study demonstrated a clear co-localization of nuclear actin with RNAP II in HeLa cells. Meanwhile, actin can be immunoprecipitated by anti-RNAP II antibody, indicating that they could interact with each other. Treatment of cells with α-amanitin induced the formation of actin bundle network in the nucleoplasm. Blocking of the formation of filamentous actin (F-actin) by cytochalasin B modified the distribution of actin. Although the actin content remained unchanged in resting and concanavalinA stimulated mouse lymphocytes, the actin content in the nuclei showed a progressive increase after stimulation. Furthermore, the antibody against actin blocked RNA synthesis in a eukaryotic in vitro transcription system. These observations implicate that nuclear actin interacts with RNAP II and may have function on the RNAP II-mediated transcription.

P0561 HOST CELL TROPHISM UNDERLIES SPECIES RESTRICTION OF HUMAN AND BOVINE CRYPTOSPORIDIUM PARVUM GENOTYPES

Introduction: It has been recognised recently that human cryptosporidiosis is usually caused by C. parvum genotype 1 (“human” C. parvum), which is not found in animals. Compared with C. parvum genotype II (cattle C. parvum), little is known of the biology of invasion of human restricted C. parvum genotype I. The aims of this study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection using an established in vitro model of infection and (ii) to examine the possibility that host specific cell tropism determines species restriction among C. parvum genotype I and genotype II using a novel ex vivo small intestinal primary cell model of infection. Methods: Oocysts of C. parvum genotype I and genotype II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small bowel biopsies and from bovine duodenum post mortem. Results: C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion following treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both types of C. parvum. Pre-treatment of oocysts with microtubule inhibitors (colchicine and vincristine) reduced the invasion efficiency of genotype II isolates. Primary cells were more efficiently infected than HCT-8 cells by C. parvum genotype I parasites. C. parvum genotype II invaded primary cells, regardless of human or bovine origin. In contrast, C. parvum genotype I invaded only human small bowel cells Conclusion: The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C. parvum genotype I does not enter primary bovine intestinal cells suggesting that the species restriction of this C. parvum genotype is due to host tissue tropism of the infecting isolate.

111VOLUMETRIC CHANGES OF BOVINE OOCYTES RESULTING FROM DEHYDRATION IN DISACCHARIDE SOLUTIONS

Vitrification of mammalian oocytes employs hypertonic solutions of cryoprotectant additives (CPAs), causing a rapid loss of water from the cell. This severe dehydration can result in osmotic shock. Oocytes are known to be altered by abrupt changes in osmotic pressure (1987 Cryobiology 24, 387–402), which may result in ultrastructural changes. Therefore, the objective of this study was to examine the effects of severe dehydration of mature bovine oocytes on the cytoskeletal arrangement of microtubules and actin filaments as well as the metaphase spindle prior to vitrification. In Experiment I, oocytes (n=30) obtained from Ovagenix (San Angelo, TX, USA) were sequentially exposed (5 or 6/treatment group) to increasing hypertonic solutions of two saccharides (0.15, 0.3, 0.5, 0.65, 0.99M sucrose and 0.125, 1.25, 0.35, 0.55, 0.65M trehalose) prepared in TCM-199 or M2 medium. Control oocytes (n=22) were subjected to isotonic control solutions (TCM-199 and M2 alone). Relative average volumes of oocytes were determined as previously described and then Boyle vant Hoff plots were constructed. In this study, bovine oocytes behaved similarly when exposed to increasing osmolalities of sucrose and trehalose in both media. The oocytes exhibited a linear decrease in relative volume as a function of 1/(osmotic pressure)to a minimum of approximately 32% of the isotonic volume for 0.99M sucrose in M2 medium (r2=0.9785) (Graphpad 2.04, GraphPad Software, San Diego, CA, USA). In the second experiment, partially denuded bovine oocytes (n=106) were sequentially dehydrated to determine the effect of dehydration on cytoskeletal structures and the meiotic spindle. Oocytes were suspended in solutions of 0.99M sucrose or 0.65M trehalose in either TCM-199 or M2 medium (concentration of sucrose and trehalose where maximum dehydration occurred in the first experiment). Correspondingly, similar oocytes (n=30) were allotted to either TCM-199 or M2 medium control groups. Oocytes were subsequently fixed, immunolabelled for tubulin (rat anti-α-yeast tubulin and anti-rat FITC) and actin (rhodamine phalloidin), stained for DNA (Hoechst 33342), and then mounted for analysis. Cytoskeletal arrangement and DNA localization were visualized using fluorescence and laser-scanning microscopy. Microtubules in control oocytes were intimately associated with the meiotic spindle, which appeared as a symmetrical, barrel-shaped structure with anastral poles. Intact spindles were observed in 63% of the control oocytes compared with 0% in all treatment groups, with the exception of 3% of the oocytes suspended in 0.99M sucrose in TCM-199. Co-localization of actin with tubulin was observed in all oocytes with intact spindles. In conclusion, bovine oocytes respond as perfect osmometers when suspended in hypertonic solutions of saccharides. Oocytes exposed to such solutions exhibited extensive disruption of their meiotic spindles, which would undoubtedly result in reduced fertilization and abnormal development. Further study is needed to determine whether the effects of dehydration can be reversed by sequential rehydration.

Expression and immunolocalisation of neutral endopeptidase in prostate cancer.

Neutral Endopeptidase (NEP) is a cell surface enzyme that cleaves and inactivates neuropeptides. When present on androgen-dependent prostate cancer (PC) cells, NEP inactivates growth stimulatory neuropeptides. After androgen ablation NEP expression decreases and neuropeptides can enhance cell growth, leading to the development of androgen-independent, neuropeptide stimulated PC. Aim of the study was to analyse the expression, localisation and distribution of NEP in benign and malignant prostatic tissues and its relation to the cytoskeleton. Immunohistochemistry (IHC) was performed to localise NEP in fixed specimens from normal prostatic tissue, benign prostate hyperplasia (BPH) and PC of Gleason grade 2-5. In situ hybridisation and Western blotting experiments were carried out to confirm NEP gene expression and translation to mature protein in BPH and PC tissue. Confocal laser scanning microscopy was utilised to investigate whether development of high grade prostate tumours was accompanied by changes in intracellular actin/NEP colocalisation patterns. Finally, the proliferative activity in relation to loss of NEP expression was investigated by dual staining of NEP and Ki-67 in prostatic tumours. In situ hybridisation studies revealed preserved expression of NEP mRNA in epithelial cells of PC. NEP was by IHC shown to be located in the apical plasma membrane of normal epithelial cells and BPH tissue. In PC a Gleason grade dependent shift of the NEP distribution pattern towards a heterogeneous, partly cytoplasmic allocation of the protein was found. Compared to BPH tissue, specimens derived from PC showed very low IHC-staining intensity for NEP protein. In high grade PC the typical apical colocalisation of actin and NEP was lost and a strong granular cytoplasmic NEP staining was found. PC areas with a high expression of NEP displayed diminished proliferative activity i.e. low staining intensity for Ki-67. NEP is differentially expressed in the normal and the pathologically altered prostate with a clear shift from a membrane bound to a cytoplasmic distribution pattern in high-grade tumours and loss of NEP expression in areas of high proliferative activity. The data presented support an active involvement of NEP in the progression of androgen-independent PC. Further studies are needed to unravel the mechanisms underlying the cytoplasmic NEP distribution in PC.

Colocalization of synapsin and actin during synaptic vesicle recycling.

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division.

We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed.

MOESIN Crosslinks Actin and Cell Membrane in Drosophila Oocytes and Is Required for OSKAR Anchoring

In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte [1–3]. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent [4–7]. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring [4]. This communication demonstrates that mutations in the Drosophila moesin (Dmoe) gene that encodes another actin binding protein result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in failure of embryonic germ cell development. In Dmoe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. In line with the earlier observations [6, 8], colocalization of ectopic actin and OSK protein in Dmoe mutants suggests that the actin cytoskeleton anchors OSK protein to the subcortical cytoplasmic area of the Drosophila oocyte.

Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells.

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-delta, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-delta were verified by Western blot analysis of immunoprecipitated proteins. PKC-delta was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-delta. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-delta. Binding of recombinant PKC-delta to NKCC1 was not detected in overlay assays. Rather, activated PKC-delta bound to actin, and this interaction was prevented by a peptide encoding deltaC2, a C2-like domain based on the amino acid sequence of PKC-delta. deltaC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-delta in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-delta indicated colocalization of PKC-delta and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-delta.

The large GTPase dynamin regulates actin comet formation and movement in living cells.

The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2DeltaPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.

Dystrophin isoform Dp71 is present in lamellipodia and focal complexes in human astrocytoma cells U-373 MG

Dp71 is the most abundant product of the dmd gene in the brain. There are at least 2 isoforms derived from alternative splicing of exon 78 (Dp71d, which contains exon 78 and Dp71f, the spliced isoform) but the precise localization and function of each isoform is still unknown. In the present study, we demonstrate by RT-PCR that the Dp71f isoform is present in an astrocytoma cell line U-373 MG, and its subcellular localization was determined in the cytoplasm, particularly in perinuclear areas, with lower amounts towards the periphery but increasing in the leader borders of lamellipodia and focal complexes. Double labeling indirect immunofluorescence showed that Dp71f colocalized with actin-like beta-dystroglycan and beta-1 integrin. We also demonstrated by triple labeling that Dp71f was colocalized with actin and two members of integrin complexes, alpha-actinin and vinculin, in focal complexes. Ventral plasma membranes were enriched and in those containing focal complex proteins, we found colocalization of Dp71f, actin and vinculin. It is concluded that U-373 MG cells express Dp71f as part of lamellipodia and focal complex proteins, and possibly connected via distroglycan complexes to integrin complexes.

Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins.