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2.  XPC promotes MDM2-mediated degradation of the p53 tumor suppressor 
Molecular Biology of the Cell  2014;25(2):213-221.
XPC binds MDM2 ubiquitin ligase and participates in the MDM2-mediated p53 degradation. Furthermore, XPC overexpression stimulates p53 degradation following UV irradiation. Combined, the results suggest a key role of XPC in p53 degradation.
Although ubiquitin receptor Rad23 has been implicated in bringing ubiquitylated p53 to the proteasome, how Rad23 recognizes p53 remains unclear. We demonstrate that XPC, a Rad23-binding protein, regulates p53 turnover. p53 protein in XPC-deficient cells remains ubiquitylated, but its association with the proteasome is drastically reduced, indicating that XPC regulates a postubiquitylation event. Furthermore, we found that XPC participates in the MDM2-mediated p53 degradation pathway via direct interaction with MDM2. XPC W690S pathogenic mutant is specifically defective for MDM2 binding and p53 degradation. p53 is known to become stabilized following UV irradiation but can be rendered unstable by XPC overexpression, underscoring a critical role of XPC in p53 regulation. Elucidation of the proteolytic role of XPC in cancer cells will help to unravel the detailed mechanisms underlying the coordination of DNA repair and proteolysis.
PMCID: PMC3890342  PMID: 24258024
3.  Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data 
Molecular Biology of the Cell  2014;25(2):222-233.
To better define how cell wall structure affects morphogenesis, the morphology of yeast cells was analyzed quantitatively after treatment with the three drugs that inhibit different aspects of cell wall synthesis. These drugs induced both similar effects, including broader necks and increased morphological variation, and distinct effects.
The cell wall of budding yeast is a rigid structure composed of multiple components. To thoroughly understand its involvement in morphogenesis, we used the image analysis software CalMorph to quantitatively analyze cell morphology after treatment with drugs that inhibit different processes during cell wall synthesis. Cells treated with cell wall–affecting drugs exhibited broader necks and increased morphological variation. Tunicamycin, which inhibits the initial step of N-glycosylation of cell wall mannoproteins, induced morphologies similar to those of strains defective in α-mannosylation. The chitin synthase inhibitor nikkomycin Z induced morphological changes similar to those of mutants defective in chitin transglycosylase, possibly due to the critical role of chitin in anchoring the β-glucan network. To define the mode of action of echinocandin B, a 1,3-β-glucan synthase inhibitor, we compared the morphology it induced with mutants of Fks1 that contains the catalytic domain for 1,3-β-glucan synthesis. Echinocandin B exerted morphological effects similar to those observed in some fks1 mutants, with defects in cell polarity and reduced glucan synthesis activity, suggesting that echinocandin B affects not only 1,3-β-glucan synthesis, but also another functional domain. Thus our multivariate analyses reveal discrete functions of cell wall components and increase our understanding of the pharmacology of antifungal drugs.
PMCID: PMC3890343  PMID: 24258022
4.  Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development 
Molecular Biology of the Cell  2014;25(2):234-244.
Nel is a thrombospondin-1–like extracellular glycoprotein that is predominantly expressed in the vertebrate nervous system. It stimulates the genesis of retinal ganglion cells (RGCs) by promoting their differentiation and survival during development and is essential for production of proper numbers of RGCs.
For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.
PMCID: PMC3890344  PMID: 24258025
5.  ARP2/3-mediated junction-associated lamellipodia control VE-cadherin–based cell junction dynamics and maintain monolayer integrity 
Molecular Biology of the Cell  2014;25(2):245-256.
The ARP2/3 complex controls junction-associated intermittent lamellipodia (JAIL), which trigger VE-cadherin adhesion and dynamics. JAIL formation maintains paraendothelial barrier function under physiological conditions and depends on the local VE-cadherin concentration.
Maintenance and remodeling of endothelial cell junctions critically depend on the VE-cadherin/catenin complex and its interaction with the actin filament cytoskeleton. Here we demonstrate that local lack of vascular endothelial (VE)-cadherin at established cell junctions causes actin-driven and actin-related protein 2/3 complex (ARP2/3)–controlled lamellipodia to appear intermittently at those sites. Lamellipodia overlap the VE-cadherin–free adjacent plasma membranes and facilitate formation of new VE-cadherin adhesion sites, which quickly move into the junctions, driving VE-cadherin dynamics and remodeling. Inhibition of the ARP2/3 complex by expression of the N-WASP (V)CA domain or application of two ARP2/3 inhibitors, CK-548 and CK-666, blocks VE-cadherin dynamics and causes intercellular gaps. Furthermore, expression of carboxy-terminal–truncated VE-cadherin increases the number of ARP2/3-controlled lamellipodia, whereas overexpression of wild-type VE-cadherin largely blocks it and decreases cell motility. The data demonstrate a functional interrelationship between VE-cadherin–mediated cell adhesion and actin-driven, ARP2/3-controlled formation of new VE-cadherin adhesion sites via intermittently appearing lamellipodia at established cell junctions. This coordinated mechanism controls VE-cadherin dynamics and cell motility and maintains monolayer integrity, thus potentially being relevant in disease and angiogenesis.
PMCID: PMC3890345  PMID: 24227887
6.  Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure 
Molecular Biology of the Cell  2014;25(2):257-266.
Acetylation of α-tubulin is uniquely located on the microtubule lumen and away from most MAPs. It does not affect microtubule structure and must affect only proteins that bind directly to the lumen. αTAT1 can interact with the tubulin C-termini, which could facilitate access to its luminal site and make it responsive to other modifications.
Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.
PMCID: PMC3890346  PMID: 24227885
7.  Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division 
Molecular Biology of the Cell  2014;25(2):267-275.
The midbody ring (MR) is asymmetrically segregated during asymmetric divisions of germline stem cells (GSCs) in Drosophila. Male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which inherit the daughter centrosome, inherit the MR. Moreover, stem cell identity correlates with the mode of MR inheritance.
Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. Here we show that the MR is asymmetrically segregated during GSC divisions in a centrosome age–dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which we here show inherit the daughter centrosome, inherit the MR. We further show that stem cell identity correlates with the mode of MR inheritance. Together our data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors.
PMCID: PMC3890347  PMID: 24227883
8.  ESCRT regulates surface expression of the Kir2.1 potassium channel 
Molecular Biology of the Cell  2014;25(2):276-289.
The Kir2.1 potassium channel is targeted by endoplasmic reticulum–associated degradation in yeast. To identify other Kir2.1 quality control factors, a novel yeast screen was performed. ESCRT components were among the strongest hits from the screen. Consistent with these data, ESCRT also regulates Kir2.1 stability in human cells.
Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum–associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.
PMCID: PMC3890348  PMID: 24227888
9.  Lipid droplet autophagy in the yeast Saccharomyces cerevisiae 
Molecular Biology of the Cell  2014;25(2):290-301.
Lipid droplet formation and degradation are pivotal processes in preventing lipotoxicity and providing energy sources and signaling molecules. This is the first demonstration of lipid droplet turnover in yeast by microautophagy. Lipophagy is distinct from ER-phagy, mitophagy, and pexophagy and contributes to neutral lipid homeostasis by vacuolar lipolysis.
Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.
PMCID: PMC3890349  PMID: 24258026
10.  Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells 
Molecular Biology of the Cell  2014;25(2):302-317.
The Smc5/6 complex plays a critical role in processing DNA replication and is indispensable for sister chromatid assembly and faithful segregation in mitosis.
The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.
PMCID: PMC3890350  PMID: 24258023
11.  Cadherin-6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural crest 
Molecular Biology of the Cell  2014;25(1):41-54.
Cadherin-6B is rapidly depleted from premigratory neural crest cells during EMT, suggestive of posttranslational mechanisms. ADAM10, ADAM19, and γ-secretase cleave cadherin-6B, and ADAM perturbation alters the premigratory neural crest cell domain. The study provides the first evidence for cadherin-6B proteolysis in neural crest cells during EMT.
The epithelial-to-mesenchymal transition (EMT) is a highly coordinated process underlying both development and disease. Premigratory neural crest cells undergo EMT, migrate away from the neural tube, and differentiate into diverse cell types during vertebrate embryogenesis. Adherens junction disassembly within premigratory neural crest cells is one component of EMT and, in chick cranial neural crest cells, involves cadherin-6B (Cad6B) down-regulation. Whereas Cad6B transcription is repressed by Snail2, the rapid loss of Cad6B protein during EMT is suggestive of posttranslational mechanisms that promote Cad6B turnover. For the first time in vivo, we demonstrate Cad6B proteolysis during neural crest cell EMT, which generates a Cad6B N-terminal fragment (NTF) and two C-terminal fragments (CTF1/2). Coexpression of relevant proteases with Cad6B in vitro shows that a disintegrin and metalloproteinases (ADAMs) ADAM10 and ADAM19, together with γ-secretase, cleave Cad6B to produce the NTF and CTFs previously observed in vivo. Of importance, both ADAMs and γ-secretase are expressed in the appropriate spatiotemporal pattern in vivo to proteolytically process Cad6B. Overexpression or depletion of either ADAM within premigratory neural crest cells prematurely reduces or maintains Cad6B, respectively. Collectively these results suggest a dual mechanism for Cad6B proteolysis involving two ADAMs, along with γ-secretase, during cranial neural crest cell EMT.
PMCID: PMC3873892  PMID: 24196837
12.  A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1 
Molecular Biology of the Cell  2014;25(1):1-16.
Here we show that the Slx5/Slx8 STUbL complex is involved in the efficient degradation of the nuclear pool of Siz1, a SUMO E3 ligase with many nuclear and cytosolic substrates. This novel finding suggests that STUbLs can regulate cellular SUMO homeostasis by targeting SUMO E3 ligases.
The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.
PMCID: PMC3873881  PMID: 24196836
13.  A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9 
Molecular Biology of the Cell  2014;25(1):107-117.
A novel axonemal protein, FAP234, of Chlamydomonas is found to form a complex with a tubulin-polyglutamylating enzyme, TTLL9, and function in the stabilization and intraflagellar transport of TTLL9. These proteins are conserved in most ciliated organisms and may be specialized for regulation of ciliary motility.
Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.
PMCID: PMC3873882  PMID: 24196831
14.  Autophagosomes contribute to intracellular lipid distribution in enterocytes 
Molecular Biology of the Cell  2014;25(1):118-132.
Delivery of alimentary lipids induces immediate autophagic response in enterocytes. Forming autophagosomes are recruited to the ER membrane, where they capture nascent lipid droplets and later fuse with lysosomes, illustrating for the first time the role of autophagy in neutral-lipid distribution in enterocytes.
Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.
PMCID: PMC3873883  PMID: 24173715
15.  Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization 
Molecular Biology of the Cell  2014;25(1):133-144.
Use of photoinactivation, cisternae-specific fluorescence recovery, and high-resolution microscopy shows that the membrane tethers GRASP65 and GRASP55 on early and late Golgi membranes, respectively, are critical to the specific, homotypic fusion of the membranes on which they reside.
Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing.
PMCID: PMC3873884  PMID: 24227884
16.  Mutations in Fis1 disrupt orderly disposal of defective mitochondria 
Molecular Biology of the Cell  2014;25(1):145-159.
The mitochondrial fission protein Drp1 binds to Mff on mitochondria, followed by entry into a complex with Fis1 at the ER–mitochondrial interface. Mutations in Fis1 disrupt disposal of defective mitochondria when fission is induced by stress. Fis1 thus acts in sequence with Mff to couple mitochondrial fission with downstream degradation processes.
Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes.
PMCID: PMC3873885  PMID: 24196833
17.  The SUMO proteases SENP1 and SENP2 play a critical role in nucleoporin homeostasis and nuclear pore complex function 
Molecular Biology of the Cell  2014;25(1):160-168.
A gap remains in the understanding of how nucleoporins are coordinately produced and assembled into macromolecular pore complexes. Here two vertebrate SUMO proteases are found to be important for proper assembly of nuclear pores and maintenance of homeostatic levels of certain nucleoporins.
Nuclear pore complexes are composed of ∼30 different proteins, each present at the pore in multiple copies. Together these proteins create specialized channels that convey cargo between the cytoplasm and the nuclear interior. With the building blocks of nuclear pores identified, one challenge is to decipher how these proteins are coordinately produced and assembled into macromolecular pore structures with each cell division. Specific individual pore proteins and protein cofactors have been probed for their role in the assembly process, as well as certain kinases that add a layer of regulation via the phosphorylation status of nucleoporins. Other posttranslational modifications are candidates for coordinating events of pore assembly as well. In this study of two pore-associated small ubiquitin-like modifier (SUMO) proteases, sentrin/SUMO-specific protease 1 (SENP1) and SENP2, we observe that many nucleoporins are mislocalized and, in some cases, reduced in level when SENP1 and SENP2 are codepleted. The pore complexes present under these conditions are still capable of transport, although the kinetics of specific cargo is altered. These results reveal a new role for the pore-associated SENPs in nucleoporin homeostasis and in achieving proper configuration of the nuclear pore complex.
PMCID: PMC3873886  PMID: 24196834
18.  NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies 
Molecular Biology of the Cell  2014;25(1):169-183.
Paraspeckles are subnuclear structures formed around NEAT1 lncRNA. Paraspeckles became enlarged after proteasome inhibition caused by NEAT1 transcriptional activation, leading to protein sequestration into paraspeckles. The NEAT1-dependent sequestration affects the transcription of several genes, arguing for a novel role for lncRNA in gene regulation.
Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1−/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.
PMCID: PMC3873887  PMID: 24173718
19.  ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion 
Molecular Biology of the Cell  2014;25(1):17-29.
The small GTPase ARF1 is overexpressed in invasive breast cancer cells. This ARF isoform controls MMP-9 activity to degrade the extracellular matrix by regulating invadopodia maturation and microvesicle shedding. The molecular mechanisms by which ARF1 controls invasiveness involve regulation of the Rho/MLC pathway.
Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.
PMCID: PMC3873888  PMID: 24196838
20.  PDLIM2 regulates transcription factor activity in epithelial-to-mesenchymal transition via the COP9 signalosome 
Molecular Biology of the Cell  2014;25(1):184-195.
PDLIM2 integrates cytoskeletal signaling with gene expression to enable reversible differentiation of epithelial cancer cells. PDLIM2 associates with the COP9 signalosome and controls its nuclear translocation and the stability of key transcription factors necessary for either a mesenchymal or an epithelial phenotype.
Epithelial cell differentiation and polarized migration associated with epithelial-to-mesenchymal transition (EMT) in cancer requires integration of gene expression with cytoskeletal dynamics. Here we show that the PDZ-LIM domain protein PDLIM2 (Mystique/SLIM), a known cytoskeletal protein and promoter of nuclear nuclear factor κB (NFκB) and signal transducer and activator of transcription (STAT) degradation, regulates transcription factor activity and gene expression through the COP9 signalosome (CSN). Although repressed in certain cancers, PDLIM2 is highly expressed in invasive cancer cells. Here we show that PDLIM2 suppression causes loss of directional migration, inability to polarize the cytoskeleton, and reversal of the EMT phenotype. This is accompanied by altered activity of several transcription factor families, including β-catenin, Ap-1, NFκB, interferon regulatory factors, STATs, JUN, and p53. We also show that PDLIM2 associates with CSN5, and cells with suppressed PDLIM2 exhibit reduced nuclear accumulation and deneddylation activity of the CSN toward the cullin 1 and cullin 3 subunits of cullin-RING ubiquitin ligases. Thus PDLIM2 integrates cytoskeleton signaling with gene expression in epithelial differentiation by controlling the stability of key transcription factors and CSN activity.
PMCID: PMC3873889  PMID: 24196835
21.  The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2014;25(1):196-211.
Sphingolipids play crucial roles in the determination of growth and survival of eukaryotic cells. The budding yeast protein kinase Sch9 is not only an effector, but also a regulator of sphingolipid metabolism. This new function provides a crucial link between nutrient and sphingolipid signaling.
The Saccharomyces cerevisiae protein kinase Sch9 is an in vitro and in vivo effector of sphingolipid signaling. This study examines the link between Sch9 and sphingolipid metabolism in S. cerevisiae in vivo based on the observation that the sch9Δ mutant displays altered sensitivity to different inhibitors of sphingolipid metabolism, namely myriocin and aureobasidin A. Sphingolipid profiling indicates that sch9Δ cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the target of rapamycin complex 1–Sch9 signaling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby revealing, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. In addition, we establish that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. Given that sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism.
PMCID: PMC3873890  PMID: 24196832
22.  Persistent telomere cohesion triggers a prolonged anaphase 
Molecular Biology of the Cell  2014;25(1):30-40.
Telomeres use distinct mechanisms to mediate cohesion between sister chromatids. However, the motivation for a specialized mechanism is not well understood. Fluorescence in situ hybridization and live-cell imaging show that persistent sister chromatid cohesion at telomeres triggers a prolonged anaphase in normal human cells and cancer cells.
Telomeres use distinct mechanisms (not used by arms or centromeres) to mediate cohesion between sister chromatids. However, the motivation for a specialized mechanism at telomeres is not well understood. Here we show, using fluorescence in situ hybridization and live-cell imaging, that persistent sister chromatid cohesion at telomeres triggers a prolonged anaphase in normal human cells and cancer cells. Excess cohesion at telomeres can be induced by inhibition of tankyrase 1, a poly(ADP-ribose) polymerase that is required for resolution of telomere cohesion, or by overexpression of proteins required to establish telomere cohesion, the shelterin subunit TIN2 and the cohesin subunit SA1. Regardless of the method of induction, excess cohesion at telomeres in mitosis prevents a robust and efficient anaphase. SA1- or TIN2-induced excess cohesion and anaphase delay can be rescued by overexpression of tankyrase 1. Moreover, we show that primary fibroblasts, which accumulate excess telomere cohesion at mitosis naturally during replicative aging, undergo a similar delay in anaphase progression that can also be rescued by overexpression of tankyrase 1. Our study demonstrates that there are opposing forces that regulate telomere cohesion. The observation that cells respond to unresolved telomere cohesion by delaying (but not completely disrupting) anaphase progression suggests a mechanism for tolerating excess cohesion and maintaining telomere integrity. This attempt to deal with telomere damage may be ultimately futile for aging fibroblasts but useful for cancer cells.
PMCID: PMC3873891  PMID: 24173716
23.  Processive acceleration of actin barbed-end assembly by N-WASP 
Molecular Biology of the Cell  2014;25(1):55-65.
Clustered N-WASP binds directly to actin-filament barbed ends and can either slow individual filament growth or processively accelerate the assembly of bundled actin filaments. This novel Arp2/3-independent mechanism of N-WASP likely plays a role in invadopodia and podosome formation, in which both N-WASP and actin filaments are tightly clustered.
Neuronal Wiskott–Aldrich syndrome protein (N-WASP)–activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3, N-WASP binds actin-filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We use nanofibers coated with N-WASP WWCA domains as model cell surfaces and single-actin-filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed-end assembly. Individual barbed ends captured by WWCA domains grow at or below their diffusion-limited assembly rate. At high filament densities, however, overlapping filaments form buckles between their nanofiber tethers and myosin attachment points. These buckles grew ∼3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native polyproline (PP) region show similar rate enhancements in the absence of profilin, but profilin slows barbed-end acceleration from constructs containing the PP region. Increasing Mg2+ to enhance filament bundling increases the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed-end bundling. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion.
PMCID: PMC3873893  PMID: 24227886
24.  Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables 
Molecular Biology of the Cell  2014;25(1):66-75.
Fission yeast tropomyosin targets myosin-V to actin cables by favoring processivity of the motor. Live-cell imaging is used to estimate the number of myosin-V molecules per motile particle in vivo. In vitro reconstitution demonstrates the physiological relevance of tropomyosin-based targeting of this motor.
A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.
PMCID: PMC3873894  PMID: 24196839
25.  A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death 
Molecular Biology of the Cell  2014;25(1):76-86.
Interindividual variation was screened for inflammatory cell death—pyroptosis. Natural variation in expression of the tubulin isoform TUBB6 or experimental manipulation of expression altered microtubule stability and susceptibility of cells to pyroptosis. Diversity in microtubule stability regulates pyroptosis and likely other human traits.
Pyroptosis is proinflammatory cell death that occurs in response to certain microbes. Activation of the protease caspase-1 by molecular platforms called inflammasomes is required for pyroptosis. We performed a cellular genome-wide association study (GWAS) using Salmonella typhimurium infection of human lymphoblastoid cell lines as a means of dissecting the genetic architecture of susceptibility to pyroptosis and identifying unknown regulatory mechanisms. Cellular GWAS revealed that a common human genetic difference that regulates pyroptosis also alters microtubule stability. An intergenic single-nucleotide polymorphism on chromosome 18 is associated with decreased pyroptosis and increased expression of TUBB6 (tubulin, β 6 class V). TUBB6 is unique among tubulin isoforms in that its overexpression can completely disrupt the microtubule network. Cells from individuals with higher levels of TUBB6 expression have lower microtubule stability and less pyroptosis. Reducing TUBB6 expression or stabilizing microtubules pharmacologically with paclitaxel (Taxol) increases pyroptosis without affecting the other major readout of caspase-1 activation, interleukin-1β secretion. The results reveal a new role for microtubules and possibly specific tubulin isoforms in the execution of pyroptosis. Furthermore, the finding that there is common diversity in TUBB6 expression and microtubule stability could have broad consequences for other microtubule-dependent phenotypes, diseases, and pharmacological responses.
PMCID: PMC3873895  PMID: 24173717

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