RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

Homology-dependent Gene Silencing in Paramecium

Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.

Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G1 Arrest in Fission Yeast

The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p.

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36M3R cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.

Rho-dependent Regulation of Cell Spreading by the Tetraspan Membrane Protein Gas3/PMP22

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid–induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

Identification of TER94, an AAA ATPase Protein, as a Bam-dependent Component of the Drosophila Fusome

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, we have begun to identify other proteins in this network of fusome tubules. In this article we present evidence that the fly homologue of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. We also show that fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis.