Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Fission yeast Cyk3p is a transglutaminase-like protein that participates in cytokinesis and cell morphogenesis

Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.     

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

Background

For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.

Results

Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.

Conclusions

In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.     

PinX1 is recruited to the mitotic chromosome periphery by Nucleolin and facilitates chromosome congression

Mitotic chromosome movements are orchestrated by interactions between spindle microtubules and chromosomes. It is well known that kinetochore is the major site where microtubule-chromosome attachment occurs. However, the functions of other domains of chromosome such as chromosome periphery have remained elusive. Our previous studies show that PinX1 distributes to chromosome periphery and kinetochore during mitosis, and harbors the microtubule binding activity. Here we report that PinX1 interacts with Nucleolin, a chromosome periphery protein, through its C-termini. Deconvolution microscopic analyses show PinX1 mainly co-localizes with Nucleolin at chromosome periphery in prometaphase. Moreover, depletion of Nucleolin abolishes chromosome periphery localizations of PinX1, suggesting a functional interrelationship between PinX1 and Nucleolin. Importantly, repression of PinX1 and Nucleolin abrogates chromosome segregation in real-time mitosis, validating the functional importance of PinX1-Nucleolin interaction. We propose PinX1 is recruited to chromosome periphery by Nucleolin and a complex of PinX1 and Nucleolin is essential for faithful chromosome congression.     

RNF2 is recruited by WASH to ubiquitinate AMBRA1 leading to downregulation of autophagy

WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homolog) was identified to function in endosomal sorting via Arp2/3 activation. We previously demonstrated that WASH is a new interactor of BECN1 and present in the BECN1-PIK3C3 complex with AMBRA1. The AMBRA1-DDB1-CUL4A complex is an E3 ligase for K63-linked ubiquitination of BECN1, which is required for starvation-induced autophagy. WASH suppresses autophagy by inhibition of BECN1 ubiquitination. However, how AMBRA1 is regulated during autophagy remains elusive. Here, we found that RNF2 associates with AMBRA1 to act as an E3 ligase to ubiquitinate AMBRA1 via K48 linkage. RNF2 mediates ubiquitination of AMBRA1 at lysine 45. Notably, RNF2 deficiency enhances autophagy induction. Upon autophagy induction, RNF2 potentiates AMBRA1 degradation with the help of WASH. WASH deficiency impairs the association of RNF2 with AMBRA1 to impede AMBRA1 degradation. Our findings reveal another novel layer of regulation of autophagy through WASH recruitment of RNF2 for AMBRA1 degradation leading to downregulation of autophagy.     

The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.     

Proper timing of cytokinesis is regulated by Schizosaccharomyces pombe Etd1

Cytokinesis must be initiated only after chromosomes have been segregated in anaphase and must be terminated once cleavage is completed. We show that the fission yeast protein Etd1 plays a central role in both of these processes. Etd1 activates the guanosine triphosphatase (GTPase) Spg1 to trigger signaling through the septum initiation network (SIN) pathway and onset of cytokinesis. Spg1 is activated in late anaphase when spindle elongation brings spindle pole body (SPB)–localized Spg1 into proximity with its activator Etd1 at cell tips, ensuring that cytokinesis is only initiated when the spindle is fully elongated. Spg1 is active at just one of the two SPBs during cytokinesis. When the actomyosin ring finishes constriction, the SIN triggers disappearance of Etd1 from the half of the cell with active Spg1, which then triggers Spg1 inactivation. Asymmetric activation of Spg1 is crucial for timely inactivation of the SIN. Together, these results suggest a mechanism whereby cell asymmetry is used to monitor cytoplasmic partitioning to turn off cytokinesis signaling.     

PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy

Mutations in PTEN-induced putative kinase 1 (PINK1) cause recessive form of Parkinson’s disease (PD). PINK1 acts upstream of parkin, regulating mitochondrial integrity and functions. Here, we show that PINK1 in combination with parkin results in the perinuclear mitochondrial aggregation followed by their elimination. This elimination is reduced in cells expressing PINK1 mutants with wild-type parkin. Although wild-type PINK1 localizes in aggregated mitochondria, PINK1 mutants localization remains diffuse and mitochondrial elimination is not observed. This phenomenon is not observed in autophagy-deficient cells. These results suggest that mitophagy controlled by the PINK1/parkin pathway might be associated with PD pathogenesis.

Structured summary

MINT-7557195: PINK1 (uniprotkb:Q9BXM7) physically interacts (MI:0915) with LC3 (uniprotkb:Q9GZQ8) by anti tag coimmunoprecipitation (MI:0007)MINT-7557109: LC3 (uniprotkb:Q9GZQ8) and PINK1 (uniprotkb:Q9BXM7) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557121: tom20 (uniprotkb:Q15388) and PINK1 (uniprotkb:Q9BXM7) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557138: parkin (uniprotkb:O60260), PINK1 (uniprotkb:Q9BXM7) and tom20 (uniprotkb:Q15388) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557173: LC3 (uniprotkb:Q9GZQ8) physically interacts (MI:0915) with PINK1 (uniprotkb:Q9BXM7) by anti bait coimmunoprecipitation (MI:0006)     

Cyclin E is recruited to the nuclear matrix during differentiation, but is not recruited in cancer cells

Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.     

Phosphorylation of Ataxin-10 by polo-like kinase 1 is required for cytokinesis

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder,whose symptoms include cerebellar ataxia and epilepsy. The disease is caused by ATTCT expansion in the ATXN10 gene, which encodes the Ataxin-10 protein. Here we identified polo-like kinase 1 (Plk1) as one of Ataxin-10's binding partners. We show that epitope-tagged Ataxin-10 and Plk1 coimmunoprecipitate, and Plk1 phosphorylates Ataxin-10 at S77 and T82 in vitro. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation, which are rescued by wild-type Ataxin-10, but not the phosphor-deficient 2A mutant. Phosphorylation-specific antibodies towards pS77 detect specific signals at the midbody. Like the knockdown, overexpression of the 2A mutant generates multinucleated cells and the 2A mutant shows decreased interaction with the Plk1 polo-box domain. In addition, we found that Ataxin-10 is ubiquitinated, and is subject to proteasome-dependent degradation, which is delayed in the 2A mutant. We propose a model in which Plk1 phosphorylation of Ataxin-10 influences its degradation and cytokinesis, which may provide mechanistic insight to SCA10's pathogenesis.     

The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions

In addition to its well known role in targeting proteins for proteasomal degradation, ubiquitin (Ub) is also involved in promoting internalization of cell surface proteins into the endocytic pathway. Moreover, putative Ub interaction motifs (UIMs) as well as Ub-associated (UBA) domains have been identified in key yeast endocytic proteins (the epsins Ent1 and Ent2, and the Eps15 homolog Ede1). In this study, we characterized the interaction of Ub with the Ede1 UBA domain and with the UIMs of Ent1. Our data suggest that the UIMs and the UBA are involved in binding these proteins to biological membranes. We also show that the Ent1 ENTH domain binds to phosphoinositides in vitro and that Ent1 NPF motifs interact with the EH domain-containing proteins Ede1 and Pan1. Our findings indicate that the ENTH domain interaction with membrane lipids cooperates with the binding of membrane-associated Ub moieties. These events may in turn favor the occurrence of other interactions, for instance EH-NPF recognition, thus stabilizing networks of low affinity binding partners at endocytic sites.     

SHCBP1 is required for midbody organization and cytokinesis completion

The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. In this report, we analyzed the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, we demonstrate that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, we demonstrate that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. Our study gives novel insight into the role of SHCBP in cytokinesis completion.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

AlphaB-crystallin phosphorylated at Ser-59 is localized in centrosomes and midbodies during mitosis

We have reported that the three serine residues in alphaB-crystallin are phosphorylated under various stress conditions. We prepared affinity-purified antibodies recognizing each of the phosphorylated serine residues (Ser-19, Ser-45, and Ser-59, respectively) in alphaB-crystallin with peptides (p19S, p45S, or p59S) that contained the corresponding phosphorylated serine residue. Immunocytochemically anti-p45S antibodies stained the cytoplasm of mitotic cells (J. Biol. Chem. 273, 28,346-28,354). We have now found that the anti-p59S antibodies recognize centrosomes and midbodies of dividing cells. alphaB-Crystallin was the only protein recognized by the anti-p59S antibodies in Western blot analyses of isolated centrosome fractions. alphaB-Crystallin phosphorylated at Ser-59 was localized at the microtubule organizing centers by means of double staining with anti-beta-tubulin antibody in aster formation analysis and was co-localized with gamma-tubulin in centrosomes. Gamma-Tubulin was co-immunoprecipitated with alphaB-crystallin in U373 glioma cell extracts. On the other hand, the location of the phosphorylated alphaB-crystallin deviated from that of alpha-tubulin or gamma-tubulin in the midbody region. Taken together with the evidences that several chaperones are distributed to centrosomes, these results suggest that alphaB-crystallin as a chaperone might be also involved in the quality control of proteins.