bluuebunny/crossref_metadata_2025 huggingface dataset:
TheDictyosteliumLIM Domain-containing Protein LIM2 Is Essential for Proper Chemotaxis and Morphogenesis
Authors: Chien Sharon , Chung Chang Y. , Sukumaran Sujatha , Osborne Nicholas , Lee Susan , Ellsworth Charlene , McNally James G. , Firtel Richard A.
Published: July 2013

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin–enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell–cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of thelimB null strain, suggesting that thelimB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.
AZU-1: A Candidate Breast Tumor Suppressor and Biomarker for Tumor Progression
Authors: Chen Huei-Mei , Schmeichel Karen L. , Mian I. Saira , Lelièvre Sophie , Petersen Ole W. , Bissell Mina J.
Published: July 2013

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.
A Proline-rich Region and Nearby Cysteine Residues Target XLαs to the Golgi Complex Region
Authors: Ugur Ozlem , Jones Teresa L. Z.
Published: July 2013

XLαs is a splice variant of the heterotrimeric G protein, Gαs, found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLαs for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLαs incorporated [3H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLαs without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Gαi2splice variant, dynamin II, βIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein α subunits and possibly for other peripheral membrane proteins as well.
The Autocrine Mitogenic Loop of the CiliateEuplotes raikovi: The Pheromone Membrane-bound Forms Are the Cell Binding Sites and Potential Signaling Receptors of Soluble Pheromones
Authors: Ortenzi Claudio , Alimenti Claudio , Vallesi Adriana , Di Pretoro Barbara , Terza Antonietta La , Luporini Pierangelo
Published: July 2013

Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding.
Response ofXenopusCds1 in Cell-free Extracts to DNA Templates with Double-stranded Ends
Authors: Guo Zijian , Dunphy William G.
Published: July 2013

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that XenopusCds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-freeXenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast,Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.
Mitotic Regulation of the APC Activator Proteins CDC20 and CDH1
Authors: Kramer Edgar R. , Scheuringer Nadja , Podtelejnikov Alexandre V. , Mann Matthias , Peters Jan-Michael
Published: July 2013

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex (APC) or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. Here, we show that CDC20 can only bind to and activate the mitotically phosphorylated form of theXenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, we propose that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APCCDC20activation. We further show that CDH1 is phosphorylated in vivo during S, G2, and M phase and that CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APCCDC20and APCCDH1; the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred.
Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck
Authors: Bijlmakers Marie-José J. E. , Marsh Mark
Published: July 2013

Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckΔSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30–45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.
Activating Phosphorylation of theSaccharomyces cerevisiaeCyclin-dependent Kinase, Cdc28p, Precedes Cyclin Binding
Authors: Ross Karen E. , Kaldis Philipp , Solomon Mark J.
Published: July 2013

Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiaeCdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28pT169Amutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.
Role of Actin and Myo2p in Polarized Secretion and Growth ofSaccharomyces cerevisiae
Authors: Karpova Tatiana S. , Reck-Peterson Samara L. , Elkind N. Barry , Mooseker Mark S. , Novick Peter J. , Cooper John A.
Published: July 2013

We examined the role of the actin cytoskeleton in secretion inSaccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant,bee1Δ(las17Δ), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Δ, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p,myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.
Complex Regulation of the Yeast Heat Shock Transcription Factor
Authors: Bonner J. José , Carlson Tage , Fackenthal Donna L. , Paddock David , Storey Kimberly , Lea Kristi
Published: July 2013

The yeast heat shock transcription factor (HSF) is regulated by posttranslational modification. Heat and superoxide can induce the conformational change associated with the heat shock response. Interaction between HSF and the chaperone hsp70 is also thought to play a role in HSF regulation. Here, we show that the Ssb1/2p member of the hsp70 family can form a stable, ATP-sensitive complex with HSF—a surprising finding because Ssb1/2p is not induced by heat shock. Phosphorylation and the assembly of HSF into larger, ATP-sensitive complexes both occur when HSF activity decreases, whether during adaptation to a raised temperature or during growth at low glucose concentrations. These larger HSF complexes also form during recovery from heat shock. However, if HSF is assembled into ATP-sensitive complexes (during growth at a low glucose concentration), heat shock does not stimulate the dissociation of the complexes. Nor does induction of the conformational change induce their dissociation. Modulation of the in vivo concentrations of the SSA and SSB proteins by deletion or overexpression affects HSF activity in a manner that is consistent with these findings and suggests the model that the SSA and SSB proteins perform distinct roles in the regulation of HSF activity.
The Yeast Heat Shock Transcription Factor Changes Conformation in Response to Superoxide and Temperature
Authors: Lee Sengyong , Carlson Tage , Christian Noah , Lea Kristi , Kedzie Jennifer , Reilly James P. , Bonner J. José
Published: July 2013

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide.
Intercellular Calcium Waves in HeLa Cells Expressing GFP-labeled Connexin 43, 32, or 26
Authors: Paemeleire Koen , Martin Patricia E. M. , Coleman Sharon L. , Fogarty Kevin E. , Carrington Walter A. , Leybaert Luc , Tuft Richard A. , Evans W. Howard , Sanderson Michael J.
Published: July 2013

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca2+]iassociated with Ca2+waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+]ichanges were characterized by initiating Ca2+puffs associated with the perinuclear ER. By contrast, in Cx–GFP-transfected cells and in the presence of apyrase, [Ca2+]ichanges were propagated without initiating perinuclear Ca2+puffs and were communicated between cells at the sites of the Cx–GFP gap junctions. The efficiency of Cx expression determined the extent of Ca2+wave propagation. These results demonstrate that intercellular Ca2+waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.
The Tetraspanin CD63/lamp3 Cycles between Endocytic and Secretory Compartments in Human Endothelial Cells
Authors: Kobayashi Toshihide , Vischer Ulrich M. , Rosnoblet Corinne , Lebrand Cécile , Lindsay Margaret , Parton Robert G. , Kruithof Egbert K. O. , Gruenberg Jean
Published: July 2013

In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular–multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel–Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel–Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel–Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel–Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel–Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel–Palade bodies.
Inositol 1,4,5-Trisphosphate Directs Ca2+Flow between Mitochondria and the Endoplasmic/Sarcoplasmic Reticulum: A Role in Regulating Cardiac Autonomic Ca2+Spiking
Authors: Jaconi Marisa , Bony Claire , Richards Stephen M. , Terzic André , Arnaudeau Serge , Vassort Guy , Pucéat Michel
Published: July 2013

The signaling role of the Ca2+releaser inositol 1,4,5-trisphosphate (IP3) has been associated with diverse cell functions. Yet, the physiological significance of IP3in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP3generated by photolysis of caged IP3or by purinergic activation of phospholipase Cγ slowed down or abolished autonomic Ca2+spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cγ antibody prevented the IP3-mediated purinergic effect. IP3triggered a ryanodine- and caffeine-insensitive Ca2+release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca2+and potential, IP3induced transient Ca2+loading and depolarization of the organelles. These mitochondrial changes were associated with Ca2+depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca2+spiking. Thus, IP3acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP3in excitable cells, the regulation of cardiac autonomic activity.
A Novel Selection Regime for Differentiation Defects Demonstrates an Essential Role for the Stumpy Form in the Life Cycle of the African Trypanosome
Authors: Tasker Maria , Wilson Judith , Sarkar Mitali , Hendriks Ed , Matthews Keith
Published: July 2013

A novel selection scheme has been developed to isolate bloodstream forms of Trypanosoma brucei, which are defective in their ability to differentiate to the procyclic stage. Detailed characterization of one selected cell line (defective in differentiation clone 1 [DiD-1]) has demonstrated that these cells are indistinguishable from the wild-type population in terms of their morphology, cell cycle progression, and biochemical characteristics but are defective in their ability to initiate differentiation to the procyclic form. Although a small proportion of DiD-1 cells remain able to transform, deletion of the genes for glycophosphatidyl inositol-phospholipase C demonstrated that this enzyme was not responsible for this inefficient differentiation. However, the attenuated growth of the Δ-glycophosphatidyl inositol-phospholipase C DiD-1 cells in mice permitted the expression of stumpy characteristics in this previously monomorphic cell line, and concomitantly their ability to differentiate efficiently was restored. Our results indicate that monomorphic cells retain expression of a characteristic of the stumpy form essential for differentiation, and that this is reduced in the defective cells. This approach provides a new route to dissection of the cytological and molecular basis of life cycle progression in the African trypanosome.
Intracellular Transport, Assembly, and Degradation of Wild-Type and Disease-linked Mutant Gap Junction Proteins
Authors: VanSlyke Judy K. , Deschenes Suzanne M. , Musil Linda S.
Published: July 2013

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t1/2< 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.
α5β1 Integrin Protects Intestinal Epithelial Cells from Apoptosis through a Phosphatidylinositol 3-Kinase and Protein Kinase B–dependent Pathway
Authors: Lee Jung Weon , Juliano R.L.
Published: July 2013

Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differentiation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. Here we examine the role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin α5 subunit in RIE1 cells conferred protection against several proapoptotic stimuli. In contrast, overexpression of the integrin α2 subunit had no effect on cell survival. The antiapoptotic effect of the α5 subunit was partially retained by a mutated version that had a truncation of the cytoplasmic domain. The antiapoptotic effects of the full-length or truncated α5 subunit were reversed upon treatment with inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase), suggesting that the α5β1 integrin might interact with the PI-3-kinase/Akt survival pathway. When cells overexpressing α5 were allowed to adhere to fibronectin, there was a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect was seen in α2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing α5 and adhering to fibronectin, there was a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this was not seen in cells overexpressing α2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocked to ability of α5 to enhance cell survival. Thus, the α5β1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway.
Identification of a Novel Family of Nonclassic Yeast Phosphatidylinositol Transfer Proteins Whose Function Modulates Phospholipase D Activity and Sec14p-independent Cell Growth
Authors: Li Xinmin , Routt Sheri M. , Xie Zhigang , Cui Xiaoxia , Fang Min , Kearns Melissa A. , Bard Martin , Kirsch Donald R. , Bankaitis Vytas A.
Published: July 2013

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24–65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet,SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate thatSFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with “bypass-Sec14p” mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.
Interaction of the τ2 Transcriptional Activation Domain of Glucocorticoid Receptor with a Novel Steroid Receptor Coactivator, Hic-5, Which Localizes to Both Focal Adhesions and the Nuclear Matrix
Authors: Yang Lan , Guerrero Jennifer , Hong Heng , DeFranco Donald B. , Stallcup Michael R.
Published: July 2013

Hic-5 (hydrogen peroxide–inducible clone-5) is a focal adhesion protein that is involved in cellular senescence. In the present study, a yeast two-hybrid screen identified Hic-5 as a protein that interacts with a region of the glucocorticoid receptor that includes a nuclear matrix–targeting signal and the τ2 transcriptional activation domain. In transiently transfected mammalian cells, overexpression of Hic-5 potentiated the activation of reporter genes by all steroid receptors, excluding the estrogen receptor. The activity of the estrogen receptor and the thyroid hormone receptor was stimulated by Hic-5 in the presence but not in the absence of coexpressed coactivator GRIP1. In biochemical fractionations and indirect immunofluorescence assays, a fraction of endogenous Hic-5 in REF-52 cells and transiently expressed Hic-5 in Cos-1 cells was associated with the nuclear matrix. The C-terminal region of Hic-5, which contains seven zinc fingers arranged in four LIM domains, was required for interaction with focal adhesions, the nuclear matrix, steroid receptors, and the τ2 domain of glucocorticoid receptor. The N-terminal region of Hic-5 possesses a transcriptional activation domain and was essential for the coactivator activity of Hic-5. Given the coexisting cytoplasmic and nuclear distributions of Hic-5 and its role in steroid receptor–mediated transcriptional activation, it is proposed that Hic-5 might transmit signals that emanate at cell attachment sites and regulate transcription factors, such as steroid receptors.
The MAL Proteolipid Is Necessary for the Overall Apical Delivery of Membrane Proteins in the Polarized Epithelial Madin–Darby Canine Kidney and Fischer Rat Thyroid Cell Lines
Authors: Martı́n-Belmonte Fernando , Puertollano Rosa , Millán Jaime , Alonso Miguel A.
Published: July 2013

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin–Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.
Spatially Regulated Recruitment of Clathrin to the Plasma Membrane during Capping and Cell Translocation
Authors: Damer Cynthia K. , O'Halloran Theresa J.
Published: July 2013

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) inDictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.
Release of Kinesin from Vesicles by hsc70 and Regulation of Fast Axonal Transport
Authors: Tsai Ming-Ying , Morfini Gerardo , Szebenyi Györgyi , Brady Scott T.
Published: July 2013

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of eitherN-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent andN-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.
Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants
Authors: Trumtel Stéphanie , Léger-Silvestre Isabelle , Gleizes Pierre-Emmanuel , Teulières Frédéric , Gas Nicole
Published: July 2013

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.
Murine Guanylate-binding Protein: Incomplete Geranylgeranyl Isoprenoid Modification of an Interferon-γ–inducible Guanosine Triphosphate-binding Protein
Authors: Stickney John T. , Buss Janice E.
Published: July 2013

Farnesylation of Ras proteins is necessary for transforming activity. Although farnesyl transferase inhibitors show promise as anticancer agents, prenylation of the most commonly mutated Ras isoform, K-Ras4B, is difficult to prevent because K-Ras4B can be alternatively modified with geranylgeranyl (C20). Little is known of the mechanisms that produce incomplete or inappropriate prenylation. Among non-Ras proteins with CaaX motifs, murine guanylate-binding protein (mGBP1) was conspicuous for its unusually low incorporation of [3H]mevalonate. Possible problems in cellular isoprenoid metabolism or prenyl transferase activity were investigated, but none that caused this defect was identified, implying that the poor labeling actually represented incomplete prenylation of mGBP1 itself. Mutagenesis indicated that the last 18 residues of mGBP1 severely limited C20 incorporation but, surprisingly, were compatible with farnesyl modification. Features leading to the expression of mutant GBPs with partial isoprenoid modification were identified. The results demonstrate that it is possible to alter a protein's prenylation state in a living cell so that graded effects of isoprenoid on function can be studied. The C20-selective impairment in prenylation also identifies mGBP1 as an important model for the study of substrate/geranylgeranyl transferase I interactions.
Differential Role of β1Cand β1AIntegrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways
Authors: Fornaro Mara , Steger Craig A. , Bennett Anton M. , Wu J. Julie , Languino Lucia R.
Published: July 2013

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1integrin subunits, β1Cand β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1Cinhibits proliferation, whereas β1Apromotes it. We investigated the ability of β1Cand β1Ato modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1Cor human β1A. The different cytodomains of either β1Cor β1Adid not affect either association with the endogenous α2, αV, and α5subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1Cshowed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1Astable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1Cby TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1Aengagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1Cstable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1Cstable cell lines was attributable to an inhibitory effect of β1Con the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1Cantiproliferative effect. These findings show that the β1Cvariant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for β1-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.
Insights into the Structural Organization of the I1 Inner Arm Dynein from a Domain Analysis of the 1β Dynein Heavy Chain
Authors: Perrone Catherine A. , Myster Steven H. , Bower Raqual , O'Toole Eileen T. , Porter Mary E.
Published: July 2013

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis.ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into theDhc10 transcription unit, which encodes the 1β Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1β Dhc fragment and a full-length 1α Dhc. Electron microscopic analysis reveals the location of the missing 1β Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1α Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.
The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress
Authors: Raitt Desmond C. , Johnson Anthony L. , Erkine Alexander M. , Makino Kozo , Morgan Brian , Gross David S. , Johnston Leland H.
Published: July 2013

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.
The Kar3p Kinesin-related Protein Forms a Novel Heterodimeric Structure with Its Associated Protein Cik1p
Authors: Barrett Jennifer G. , Manning Brendan D. , Snyder Michael
Published: July 2013

Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end–directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.
VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts
Authors: Randhawa Varinder K. , Bilan Philip J. , Khayat Zayna A. , Daneman Nicholas , Liu Zhi , Ramlal Toolsie , Volchuk Allen , Peng Xiao-Rong , Coppola Thierry , Regazzi Romano , Trimble William S. , Klip Amira
Published: July 2013

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.