Abl Kinase Inhibits the Engulfment of Apopotic Cells in Caenorhabditis elegans

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.     

Caenorhabditis elegans Myotubularin MTM-1 Negatively Regulates the Engulfment of Apoptotic Cells

During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.     

吞噬凋亡细胞的机制

被吞噬细胞吞噬是多数凋亡细胞的命运.凋亡细胞表面膜磷脂酰丝氨酸的暴露、膜碳水化合物的改变及表面糖蛋白的重新分布和聚集导致被吞噬细胞识别与摄取.吞噬细胞的多种受体参与吞噬过程,有些受体参与栓系凋亡细胞,有些激发巨吞饮的摄取机制.吞噬的摄取过程因吞噬细胞和凋亡细胞的类型差异而不同.至少有7种线虫吞噬基因及其哺乳动物同源物组成两条部分重叠而又平行的摄取信息传导通路.吞噬基因的突变可以改变凋亡细胞的进程.吞噬功能的缺陷将影响机体正常的免疫应答.     

凋亡细胞被识别和吞噬机制

凋亡细胞能被吞噬细胞吞噬,这对于正常组织的动态平衡和免疫反应是非常重要的。在凋亡细胞被吞噬（engulfment）的过程中,吞噬细胞表面存在大量的受体来识别凋亡细胞发出的信号,如：“吃我（eat-me）”信号、缺少存在于健康细胞上的“不吃我（don’t-eat-me）”信号以及由凋亡细胞分泌的可溶性“来吃我（come-get-me）”信号。至少有7种线虫（Caenorhabditis elegans）吞噬基因（它们在哺乳动物中存在同系物）组成了两条平行但部分重叠的吞噬信号通路,并且通过一个类似于巨胞CL（macropinocytosis）的“栓系-激活（tether and tickle）”保守机制吞噬凋亡细胞,这个机制因吞噬细胞和凋亡细胞的种类以及细胞凋亡后的时间差异而不同。     

Caspase Activity Is Required for Engulfment of Apoptotic Cells

Clearance of apoptotic cells by phagocytic neighbors is crucial for normal development of multicellular organisms. However, how phagocytes discriminate between healthy and dying cells remains poorly understood. We focus on glial phagocytosis of apoptotic neurons during development of the Drosophila central nervous system. We identified phosphatidylserine (PS) as a ligand on apoptotic cells for the phagocytic receptor Six Microns Under (SIMU) and report that PS alone is not sufficient for engulfment. Our data reveal that, additionally to PS exposure, caspase activity is required for clearance of apoptotic cells by phagocytes. Here we demonstrate that SIMU recognizes and binds PS on apoptotic cells through its N-terminal EMILIN (EMI), Nimrod 1 (NIM1), and NIM2 repeats, whereas the C-terminal NIM3 and NIM4 repeats control SIMU affinity to PS. Based on the structure-function analysis of SIMU, we discovered a novel mechanism of internal inhibition responsible for differential affinities of SIMU to its ligand which might prevent elimination of living cells exposing PS on their surfaces.     

Apoptotic regulators promote cytokinetic midbody degradation in C. elegans

Cell death genes are essential for apoptosis and other cellular events, but their nonapoptotic functions are not well understood. The midbody is an important cytokinetic structure required for daughter cell abscission, but its fate after cell division remains elusive in metazoans. In this paper, we show through live-imaging analysis that midbodies generated by Q cell divisions in Caenorhabditis elegans were released to the extracellular space after abscission and subsequently internalized and degraded by the phagocyte that digests apoptotic Q cell corpses. We further show that midbody degradation is defective in apoptotic cell engulfment mutants. Externalized phosphatidylserine (PS), an engulfment signal for corpse phagocytosis, exists on the outer surface of the midbody, and inhibiting PS signaling delayed midbody clearance. Thus, our findings uncover a novel function of cell death genes in midbody internalization and degradation after cell division.     

Asymmetric Neuroblast Divisions Producing Apoptotic Cells Require the Cytohesin GRP-1 in Caenorhabditis elegans

Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1’s GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function.     

The Function of a Spindle Checkpoint Gene bub-1 in C. elegans Development

Background

The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant''s incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.

Methods and Findings

In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.

Conclusions

Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

Information Flow through a Model of the C. elegans Klinotaxis Circuit

Understanding how information about external stimuli is transformed into behavior is one of the central goals of neuroscience. Here we characterize the information flow through a complete sensorimotor circuit: from stimulus, to sensory neurons, to interneurons, to motor neurons, to muscles, to motion. Specifically, we apply a recently developed framework for quantifying information flow to a previously published ensemble of models of salt klinotaxis in the nematode worm Caenorhabditis elegans. Despite large variations in the neural parameters of individual circuits, we found that the overall information flow architecture circuit is remarkably consistent across the ensemble. This suggests structural connectivity is not necessarily predictive of effective connectivity. It also suggests information flow analysis captures general principles of operation for the klinotaxis circuit. In addition, information flow analysis reveals several key principles underlying how the models operate: (1) Interneuron class AIY is responsible for integrating information about positive and negative changes in concentration, and exhibits a strong left/right information asymmetry. (2) Gap junctions play a crucial role in the transfer of information responsible for the information symmetry observed in interneuron class AIZ. (3) Neck motor neuron class SMB implements an information gating mechanism that underlies the circuit’s state-dependent response. (4) The neck carries more information about small changes in concentration than about large ones, and more information about positive changes in concentration than about negative ones. Thus, not all directions of movement are equally informative for the worm. Each of these findings corresponds to hypotheses that could potentially be tested in the worm. Knowing the results of these experiments would greatly refine our understanding of the neural circuit underlying klinotaxis.     

microRNA-mediated translation repression through GYF-1 and IFE-4 in C. elegans development

microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35–42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs.     

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL''s in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.     

HIV-1 Tat蛋白抑制骨髓间充质干细胞的造血支持功能

目的:通过研究人类免疫缺陷病毒1型(HIV-1)Tat蛋白对骨髓间充质干细胞(BMSC)造血支持功能的影响,进一步揭示HIV-1感染者造血损伤的机理。方法:原代培养BMSC,流式检测其表面标志,诱导分化鉴定其多向分化潜能;免疫磁珠分选造血干细胞(HSC),流式检测其纯度;HIV-1 Tat蛋白添加到培养基中培养20天的BMSC(BMSC_(Tat))与对照BMSC(BMSC_(Con))分别作为滋养层与HSC分6组进行共培养,随后计数各组造血细胞总数,诱导分化检测造血细胞集落形成能力;RT-PCR检测BMSC_(Tat)和BMSC_(Con)造血相关因子mRNA的表达强度,ELISA检测BMSC_(Tat)和BMSC_(Con)条件培养液中造血相关因子GM-CSF及IL-6的浓度。结果:经鉴定成功培养获得原代BMSC;免疫磁珠分选的HSC纯度可达95%以上;分6组共培养进行比较,以BMSC_(Tat)为滋养层培养的造血细胞总数及造血细胞形成的集落总数均明显少于以BMSC_(Con)为滋养层;BMSC_(Tat)的造血相关因子的mRNA的表达明显弱于BMSC_(Con),BMSC_(Tat)的条件培养液中GM-CSF和IL-6的浓度均明显低于BMSC_(Con)。结论:HIV-1 Tat蛋白对BMSC的造血支持功能有明显的抑制作用。     

KiSS1 Inhibits Growth and Invasion of Osteosarcoma Cells through Inhibition of the MAPK Pathway

As a metastasis suppressor, KiSS1 has been implicated in numerous human cancers. However, recent studies have demonstrated that KiSS1 promotes tumor growth and metastasis in breast cancer, and it is unclear about the expression and function of KiSS1 in human osteosarcoma (OS). The aim of the present study was to investigate the role and molecular mechanisms of KiSS1 in human OS. The expression of KiSS1 was assessed by immunohistochemical assay using a tissue microarray procedure in forty cases of OS tissues. A gain-of-function approach was used to observe the effects of lentiviral vector-mediated overexpression of KiSS1 (Lv-KiSS1) on the biological behaviors including proliferative activities and invasive potential of OS MG-63 cells, indicated by MTT and Transwell assays, respectively. The results showed that the expression of KiSS1 protein in OS tissues was significantly lowered compared to that in adjacent non-cancerous tissues (42.5% vs 70.0%, P=0.023), and had negative correlation with distant metastases of the tumor (P=0.019). Overexpression of KiSS1 inhibited proliferation and invasion of OS cells with the decreased expression of p38 MAPK and matrix metalloproteinase-9 (MMP-9). Taken together, our findings indicate that the decreased expression of KiSS1 is correlated with distant metastasis of OS, and KiSS1 may function as a tumor suppressor in OS cells through inhibition of the MAPK pathway, suggesting that KiSS1 may serve as a potential therapeutic target for the treatment of cancer.Key words: KiSS1, osteosarcoma, growth, invasion     

Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans

In mammals, the inheritance of mitochondrion and its DNA (mtDNA) is strictly maternal, despite the fact that a sperm can inject up to 100 functional mitochondria into the oocyte during fertilization. The mechanisms responsible for the elimination of the paternal mitochondria remain largely unknown. We report here that this paternal mitochondrial elimination process is conserved in Caenorhabditis elegans, and that the lysosomal pathway actively participates in this process. Molecular and cell biological analyses indicate that in wild-type animals paternal mitochondria and mtDNA are destroyed within two hours after fertilization. In animals with compromised lysosomes, paternal mitochondria persist until late embryonic stages. Therefore, the lysosomal pathway plays an important role in degrading paternal mitochondria introduced into the oocyte during fertilization. Our study indicates that C. elegans is an excellent animal model for understanding and dissecting this conserved biological process critical for animal development and reproduction.