A nucleolus-tethering system (NoTS) based on nucleolin2 is developed by artificially tethering a protein of interest to the nucleolus for analyzing protein–protein interactions and the initiation of nuclear bodies. The NoTS is used to demonstrate a self-organization model for the biogenesis of photobodies.
Protein–protein interactions play essential roles in regulating many biological processes. At the cellular level, many proteins form nuclear foci known as nuclear bodies in which many components interact with each other. Photobodies are nuclear bodies containing proteins for light-signaling pathways in plants. What initiates the formation of photobodies is poorly understood. Here we develop a nucleolar marker protein nucleolin2 (Nuc2)–based method called the nucleolus-tethering system (NoTS) by artificially tethering a protein of interest to the nucleolus to analyze the initiation of photobodies. A candidate initiator is evaluated by visualizing whether a protein fused with Nuc2 forms body-like structures at the periphery of the nucleolus, and other components are recruited to the de novo–formed bodies. The interaction between two proteins can also be revealed through relocation and recruitment of interacting proteins to the nucleolus. Using the NoTS, we test the interactions among components in photobodies. In addition, we demonstrate that components of photobodies such as CONSTITUTIVELY PHOTOMORPHOGENIC 1, photoreceptors, and transcription factors tethered to the nucleolus have the capacity to form body-like structures at the periphery of the nucleolus, which contain other components of photobodies, suggesting a self-organization model for the biogenesis of photobodies.
Using mouse knockout strategy, the authors uncovered a role for Borg5 in microvascular angiogenesis. In primary mouse cardiac endothelial cells, Borg5 interacts with septin cytoskeleton and colocalizes with perinuclear actomyosin fibers. The data presented suggest that Borg5 and septin regulate the actomyosin activity critical for persistent directional migration.
The microvasculature is important for vertebrate organ development and homeostasis. However, the molecular mechanism of microvascular angiogenesis remains incompletely understood. Through studying Borg5 (Binder of the Rho GTPase 5), which belongs to a family of poorly understood effector proteins of the Cdc42 GTPase, we uncover a role for Borg5 in microvascular angiogenesis. Deletion of Borg5 in mice results in defects in retinal and cardiac microvasculature as well as heart development. Borg5 promotes angiogenesis by regulating persistent directional migration of the endothelial cells (ECs). In primary mouse cardiac ECs (MCECs), Borg5 associates with septins in the perinuclear region and colocalizes with actomyosin fibers. Both Borg5 deletion and septin 7 knockdown lead to a disruption of the perinuclear actomyosin and persistent directional migration. Our findings suggest that Borg5 and septin cytoskeleton spatially control actomyosin activity to ensure persistent directional migration of MCECs and efficient microvascular angiogenesis. Our studies reported here should offer a new avenue to further investigate the functions of Borg5, septin, and actomyosin in the microvasculature in the context of development and disease.
This study revealed that ubiquitously expressed transcript (UXT)-V1 is recruited to tumor necrosis factor (TNF) receptor complex I by interacting with TNF receptor-associated factor 2. UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study uncovers UXT-V1 as a novel regulator of TNF-induced apoptosis.
Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor–associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.
Human Bre1, an E3 ligase for H2B monoubiquitination, binds p53 and enhances activator-dependent transcription. Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a tumor suppressor. Here, we show that hBre1 acts as an E3 ubiquitin ligase for Ebp1 tumor suppressor and promotes its polyubiquitination and degradation. Ebp1 is polyubiquitinated in cancer cells, which is regulated by its phosphorylation. We identified hBre1 acting as an E3 ligase for Ebp1 and increasing its polyubiquitination. Depletion of hBre1 blocks Ebp1's polyubiquitination and elevates its protein level, preventing cancer proliferation. hBre1 binds Ebp1 and suppresses its repressive effect on E2F-1. Moreover, Ebp1 protein level is substantially diminished in human cancers. It is robustly phosphorylated and localized in the nucleus of primary gliomas, correlating with hBre1 subcellular residency. Thus, hBre1 inhibits Ebp1's tumor suppressive activity through mediating its polyubiquitination and degradation.
Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-α treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-α promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-α-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.
Nerve growth factor (NGF) induces neurite outgrowth and differentiation in a process that involves NGF binding to its receptor TrkA and endocytosis of the NGF–TrkA complex into signaling endosomes. Here, we find that biogenesis of signaling endosomes requires inactivation of Rab5 to block early endosome fusion. Expression of dominant-negative Rab5 mutants enhanced NGF-mediated neurite outgrowth, whereas a constitutively active Rab5 mutant or Rabex-5 inhibited this process. Consistently, inactivation of Rab5 sustained TrkA activation on the endosomes. Furthermore, NGF treatment rapidly decreased cellular level of active Rab5-GTP, as shown by pull-down assays. This Rab5 down-regulation was mediated by RabGAP5, which was shown to associate with TrkA by coimmunoprecipitation assays. Importantly, RNA interference of RabGAP5 as well as a RabGAP5 truncation mutant containing the TrkA-binding domain blocked NGF-mediated neurite outgrowth, indicating a requirement for RabGAP5 in this process. Thus, NGF signaling down-regulates Rab5 activity via RabGAP5 to facilitate neurite outgrowth and differentiation.
Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells—they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These “progenitor cells” retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.
Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.
The atlastin GTPase couples nucleotide hydrolysis to formation of a trans cross-over dimer to catalyze homotypic endoplasmic reticulum membrane fusion. Assays that separate tethering from fusion reveal that stable trans contact for tethering depends on GTP hydrolysis. In contrast, cross-over formation is required only for the fusion step.
The membrane-anchored atlastin GTPase couples nucleotide hydrolysis to the catalysis of homotypic membrane fusion to form a branched endoplasmic reticulum network. Trans dimerization between atlastins anchored in opposing membranes, accompanied by a cross-over conformational change, is thought to draw the membranes together for fusion. Previous studies on the conformational coupling of atlastin to its GTP hydrolysis cycle have been carried out largely on atlastins lacking a membrane anchor. Consequently, whether fusion involves a discrete tethering step and, if so, the potential role of GTP hydrolysis and cross-over in tethering remain unknown. In this study, we used membrane-anchored atlastins in assays that separate tethering from fusion to dissect the requirements for each. We found that tethering depended on GTP hydrolysis, but, unlike fusion, it did not depend on cross-over. Thus GTP hydrolysis initiates stable head-domain contact in trans to tether opposing membranes, whereas cross-over formation plays a more pivotal role in powering the lipid rearrangements for fusion.
Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the GEFs and GAPs that regulate its GTP-GDP cycle. TBC1D9B is identified as a Rab11a GAP in MDCK cells, where it regulates the Rab11a-dependent basolateral-to-apical transcytotic pathway.
Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the guanine nucleotide-exchange factors and GTPase-activating proteins (GAPs) that regulate its GTP-GDP cycle. We observed that in the presence of Mg2+ (2.5 mM), TBC1D9B interacted via its Tre2-Bub2-Cdc16 (TBC) domain with Rab11a, Rab11b, and Rab4a in a nucleotide-dependent manner. However, only Rab11a was a substrate for TBC1D9B-stimulated GTP hydrolysis. At limiting Mg2+ concentrations (<0.5 mM), Rab8a was an additional substrate for this GAP. In polarized Madin–Darby canine kidney cells, endogenous TBC1D9B colocalized with Rab11a-positive recycling endosomes but less so with EEA1-positive early endosomes, transferrin-positive recycling endosomes, or late endosomes. Overexpression of TBC1D9B, but not an inactive mutant, decreased the rate of basolateral-to-apical IgA transcytosis—a Rab11a-dependent pathway—and shRNA-mediated depletion of TBC1D9B increased the rate of this process. In contrast, TBC1D9B had no effect on two Rab11a-independent pathways—basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor. Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A. We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.
In prolonged activated microglia, GSK3b-iNOS-NO–dependent glycolysis activates HDAC4 degradation, which results in attenuation of inflammatory cytokine. Glycolysis is not only required for immune cell activation but is also involved in its termination by triggering HDAC4 degradation.
Activation of the inflammatory response is accompanied by a metabolic shift to aerobic glycolysis. Here we identify histone deacetylase 4 (HDAC4) as a new component of the immunometabolic program. We show that HDAC4 is required for efficient inflammatory cytokine production activated by lipopolysaccharide (LPS). Surprisingly, prolonged LPS treatment leads to HDAC4 degradation. LPS-induced HDAC4 degradation requires active glycolysis controlled by GSK3β and inducible nitric oxide synthase (iNOS). Inhibition of GSK3β or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation. We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation. Of importance, a caspase-3–resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production. Our findings identify the GSK3β-iNOS-NO axis as a critical signaling cascade that couples inflammation to metabolic reprogramming and a glycolysis-driven negative feedback mechanism that limits inflammatory response by triggering HDAC4 degradation.
HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals via up-regulation of LRP6 expression. The results highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.
Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.
Period 2 forms a trimeric complex with p53 and Mdm2. As a result, p53’s transcriptional activity and stability are modulated in unstressed cells, ensuring that basal levels are present if a p53-mediated response is needed. These data provide evidence of cross-talk between circadian and checkpoint components, adding a level of regulation to the checkpoint.
Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.
In vitro and in vivo evidence shows that activation of HIF-1a/Twist-Bmi1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis via modulation of PI3K/Akt/Snail signaling by facilitating EMT.
Hypoxia is an important microenvironmental factor in the development of renal fibrosis; however, the underlying mechanisms are not well elucidated. Here we show that hypoxia induces Bmi1 mRNA and protein expression in human tubular epithelial cells. We further demonstrate that Bmi1 expression might be directly regulated by hypoxia-inducible factor-1a (HIF-1a) under low oxygen. Moreover, chromatin immunoprecipitation and reporter gene assay studies reveal cooperative transactivation of Bmi1 by HIF-1α and Twist. Enforced Bmi1 expression induces epithelial–mesenchymal transition (EMT), whereas silencing endogenous Bmi-1 expression reverses hypoxia-induced EMT. Up-regulation of Bmi1 leads to stabilization of Snail via modulation of PI3K/Akt signaling, whereas ablation of PI3K/Akt signaling partially rescues the phenotype of Bmi1-overexpressing cells, indicating that PI3K/Akt signaling might be a major mediator of Bmi1-induced EMT. In a rat model of obstructive nephropathy, Bmi1 expression increases in a time-dependent manner. Furthermore, we demonstrate that increased levels of Bmi1, correlated with HIF-1α and Twist, are associated with patients with chronic kidney disease. We provide in vitro and in vivo evidence that activation of HIF-1a/Twist-Bmi1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis via modulation of PI3K/Akt/Snail signaling by facilitating EMT.
PML plays a critical role in the maintenance of ROS homeostasis via a unique mechanism in which PML functions as an oxidative sensor to regulate the expression of antioxidant genes through Nrf2. PML is also indispensable for sulforaphane-mediated ROS generation, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis.
Oxidative stress is a consequence of an imbalance between reactive oxygen species (ROS) production and the ability of the cytoprotective system to detoxify the reactive intermediates. The tumor suppressor promyelocytic leukemia protein (PML) functions as a stress sensor. Loss of PML results in impaired mitochondrial complex II activity, increased ROS, and subsequent activation of nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidative pathway. We also demonstrate that sulforaphane (SFN), an antioxidant, regulates Nrf2 activity by controlling abundance and subcellular distribution of PML and that PML is essential for SFN-mediated ROS increase, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis. Taking the results together, we have uncovered a novel antioxidative mechanism by which PML regulates cellular oxidant homeostasis by controlling complex II integrity and Nrf2 activity and identified PML as an indispensable mediator of SFN activity.
The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in animal cells. NME7 possesses an intrinsic kinase activity that is involved in the stimulation of the γTuRC.
As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.
Cortexillin III, a member of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins, forms heterodimers with cortexillins I and II that bind to the GAP protein DGAP1 in vivo. Cortexillin III complexes may be negative regulators of cell growth, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.
Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.
In Caenorhabditis elegans, the lysosomal cathepsin protease CPL-1 is indispensable for clearance of apoptotic cells by playing a leading role in destruction of cell corpses in phagolysosomes.
During programmed cell death, the clearance of apoptotic cells is achieved by their phagocytosis and delivery to lysosomes for destruction in engulfing cells. However, the role of lysosomal proteases in cell corpse destruction is not understood. Here we report the identification of the lysosomal cathepsin CPL-1 as an indispensable protease for apoptotic cell removal in Caenorhabditis elegans. We find that loss of cpl-1 function leads to strong accumulation of germ cell corpses, which results from a failure in degradation rather than engulfment. CPL-1 is expressed in a variety of cell types, including engulfment cells, and its mutation does not affect the maturation of cell corpse–containing phagosomes, including phagosomal recruitment of maturation effectors and phagosome acidification. Of importance, we find that phagosomal recruitment and incorporation of CPL-1 occurs before digestion of cell corpses, which depends on factors required for phagolysosome formation. Using RNA interference, we further examine the role of other candidate lysosomal proteases in cell corpse clearance but find that they do not obviously affect this process. Collectively, these findings establish CPL-1 as the leading lysosomal protease required for elimination of apoptotic cells in C. elegans.
The 26S proteasome is ubiquitinated by proteasome-associating ubiquitination enzymes. Proteasome ubiquitination impairs proteasomal degradation and is regulated by deubiquitination, substrate binding, and cellular stress. It is proposed that in situ ubiquitination autoregulates proteasomal activity in cells.
The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome's ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.
Zebrafish have four tubulin deglutamylases: Ccp1, Ccp2, Ccp5, and Ccp6. Except for ccp1, all deglutamylase genes are expressed during ciliogenesis in zebrafish. Only loss of ccp5 induces cilia hyperglutamylation and the complete spectrum of ciliopathy phenotype. ccp5 knockdown can bypass Fleer/Ift70 or Ift88 deficiency in zebrafish to form multicilia.
Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type–specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.
Motility analysis of collectively migrating epithelial sheets reveals the interplay between cellular density and leader cell positioning throughout a collective group, as well as the functional role of leader cell constraint in directing a migrating group of cells.
During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFβ-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.
mTORC2 has been shown to be involved in cytoskeletal regulation, but the mechanisms by which this takes place are poorly understood. This study shows that PKCβII is specifically required for mTORC2-dependent activation of adenylyl cyclase 9 and back retraction during neutrophil chemotaxis to chemoattractants.
Chemotaxis is a process by which cells polarize and move up a chemical gradient through the spatiotemporal regulation of actin assembly and actomyosin contractility, which ultimately control front protrusions and back retractions. We previously demonstrated that in neutrophils, mammalian target of rapamycin complex 2 (mTORC2) is required for chemoattractant-mediated activation of adenylyl cyclase 9 (AC9), which converts ATP into cAMP and regulates back contraction through MyoII phosphorylation. Here we study the mechanism by which mTORC2 regulates neutrophil chemotaxis and AC9 activity. We show that inhibition of protein kinase CβII (PKCβII) by CPG53353 or short hairpin RNA knockdown severely inhibits chemoattractant-induced cAMP synthesis and chemotaxis in neutrophils. Remarkably, PKCβII-inhibited cells exhibit specific and severe tail retraction defects. In response to chemoattractant stimulation, phosphorylated PKCβII, but not PKCα, is transiently translocated to the plasma membrane, where it phosphorylates and activates AC9. mTORC2-mediated PKCβII phosphorylation on its turn motif, but not its hydrophobic motif, is required for membrane translocation of PKCβII. Inhibition of mTORC2 activity by Rictor knockdown not only dramatically decreases PKCβII activity, but it also strongly inhibits membrane translocation of PKCβII. Together our findings show that PKCβII is specifically required for mTORC2-dependent AC9 activation and back retraction during neutrophil chemotaxis.
Smurf2 is an E3 ubiquitin ligase that regulates TGF-β/Smad signaling and is implicated in a wide variety of cellular responses. miR-322 and miR-503 repress Smurf2 translation and thus modulate TGF-β/Smad2 signaling and intestinal epithelial homeostasis.
Smad ubiquitin regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that regulates transforming growth factor β (TGF-β)/Smad signaling and is implicated in a wide variety of cellular responses, but the exact mechanisms that control Smurf2 abundance are largely unknown. Here we identify microRNA-322 (miR-322) and miR-503 as novel factors that regulate Smurf2 expression posttranscriptionally. Both miR-322 and miR-503 interact with Smurf2 mRNA via its 3′-untranslated region (UTR) and repress Smurf2 translation but do not affect total Smurf2 mRNA levels. Studies using heterologous reporter constructs reveal a greater repressive effect of miR-322/503 through a single binding site in the Smurf2 3′-UTR, whereas point mutation of this site prevents miR-322/503–induced repression of Smurf2 translation. Increased levels of endogenous Smurf2 via antagonism of miR-322/503 inhibits TGF-β–induced Smad2 activation by increasing degradation of phosphorylated Smad2. Furthermore, the increase in Smurf2 in intestinal epithelial cells (IECs) expressing lower levels of miR-322/503 is associated with increased resistance to apoptosis, which is abolished by Smurf2 silencing. These findings indicate that miR-322/503 represses Smurf2 translation, in turn affecting intestinal epithelial homeostasis by altering TGF-β/Smad2 signaling and IEC apoptosis.
ECM softness (low stiffness comparable to soft tissues) alone is sufficient to prevent cell-to-cell adherens junction formation, up-regulate MMP secretion, promote MMP activity, and induce invadosome-like protrusion formation. Such findings suggest that cell invasion in vivo is a spontaneous cell behavior in response to ECM stiffness.
Directional mesenchymal cell invasion in vivo is understood to be a stimulated event and to be regulated by cytokines, chemokines, and types of extracellular matrix (ECM). Instead, by focusing on the cellular response to ECM stiffness, we found that soft ECM (low stiffness) itself is sufficient to prevent stable cell-to-cell adherens junction formation, up-regulate matrix metalloproteinase (MMP) secretion, promote MMP activity, and induce invadosome-like protrusion (ILP) formation. Consistently, similar ILP formation was also detected in a three-dimensional directional invasion assay in soft matrix. Primary human fibroblasts spontaneously form ILPs in a very narrow range of ECM stiffness (0.1–0.4 kPa), and such ILP formation is Src family kinase dependent. In contrast, spontaneous ILP formation in malignant cancer cells and fibrosarcoma cells occurs across a much wider range of ECM stiffness, and these tumor cell ILPs are also more prominent at lower stiffness. These findings suggest that ECM softness is a natural stimulator for cellular invasiveness.
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.