胶质细胞的细胞间通讯

星型胶质细胞虽然没有动作电位,但是可以表达多种受体和离子通道,并且以细胞内钙波传递的方式来响应各类刺激。星型胶质细胞同样可以释放多种信号分子来介导细胞间的通讯。尤为特别的是,星型胶质细胞的钙波传播和突触功能的反馈调节都需要其释放ATP才得以完成。然而,星型胶质细胞释放ATP的途径和机理还有待研究。尽管人们已经在星型胶质细胞中发现了小囊泡和大致密核心囊泡的标记物,可是用以胞吐的囊泡究竟是什么还并不清楚。作者等近期的研究成果表明,FM染料——一种被成功应用于研究神经元和其他分泌型细胞囊泡循环的染料,可以特异地标记星型胶质细胞的溶酶体,并依不同程度的刺激表现出两种不同模式的钙离子依赖性胞吐：在较低强度刺激下（ATP,谷氨酸）发生部分胞吐,而在高强度刺激下（氰化钾）则发生完全胞吐。进一步研究表明,溶酶体中含有大量ATP,并且在部分胞吐时少量释放ATP,完全胞吐时大量释放ATP,同时释放溶酶体酶。选择性地裂解星型胶质细胞的溶酶体,发现ATP释放和钙波传播都消失了。总之,星型胶质细胞的溶酶体可以通过调节性胞吐对生理和病理条件下的细胞间信号传递产生重要意义。     

内体—溶酶体运输及其细胞功能

哺乳动物细胞中,内吞作用通过质膜内陷形成囊泡来摄取外界物质,经早内体到达晚内体/溶酶体降解或经再生循环回到质膜。内体运输网络参与细胞一系列重要生命活动,如信号通路调节、细胞器发生以及胞吐作用等。近年来发现Aps、BLOCs、HOPS和ESCRTs等复合体共同参与货物由胞内体到溶酶体或溶酶体相关细胞器的运送。该文主要就这些内体—溶酶体运输系统中重要蛋白复合体的组成和功能进行综述。     

胰岛素分泌及调节的分子机制

胰岛素是机体最重要的激素之一,它调节机体的血糖稳定、促进同化代谢、调节细胞的分裂分化和生长发育.胰岛β细胞的胰岛素分泌受到营养物质、神经递质和激素的精确调控.它们的作用部位可分为改变胞内第二信使物质水平的近端调节步骤（钙依赖性）,和直接作用于胞吐分子构件的末端调节步骤（钙非依赖性）.胰岛素的胞吐过程与神经递质的释放机制类似.葡萄糖等营养物质主要通过升高胞内的ATP/ADP比率,导致ATP敏感钾通道关闭、细胞膜去极化、钙内流这一途径增加胰岛素的分泌.神经递质和部分激素通过其G蛋白偶联受体-G蛋白系统的跨膜信号转换后,影响胞内IP₃、DAG、Ca²⁺等第二信使物质水平,主要通过PKA、PKC等蛋白激酶途径,调节胰岛素的分泌.胞内单体G蛋白参与了对囊泡运输和胞吐过程的调控,G蛋白也可能直接作用于胞吐过程,在分泌过程中发挥了重要的调节作用.     

可兴奋细胞胞吐胞吞的耦联机制

调节型胞吐存在于神经元、内分泌细胞和外分泌细胞等可兴奋细胞以及免疫细胞等特化细胞中,是复杂而精确调节的分泌过程。其作用广泛,与神经信号传递、激素释放、免疫反应等重要的生理活动密切相关。调节型胞吐发生后,在分秒的时间尺度上,可兴奋性细胞快速启动与胞吐发生位置密切相关的调控型胞吞,回收细胞膜脂质和囊泡的膜蛋白,迅速清除分泌位点上由于胞吐而留下的蛋白以利于下一轮的分泌,回收并填充可释放囊泡库,维持细胞膜的平衡。该文先分别介绍可兴奋细胞中胞吐和胞吞的主要模式,然后探讨了它们之间耦联的机制。     

中国少棘蜈蚣（Scolopendra subspinipes mutillans）的毒腺结构

用免疫组化和光镜、透射电镜等观察了中国少棘蜈蚣毒腺的结构。结果显示,纵贯颚肢的弯月形毒腺为单管泡状腺,主要由柱状分泌细胞和介于其间的纤细表皮细胞组成。被肌肉束环绕的分泌细胞辐射状排列于几丁质的毒液导管外,其纤细的颈部由环状括约肌控制,分泌端以折叠回转的单向瓣膜经导管壁上的孔道直接伸入管腔,膨大的盲端直达毒腺底膜。高电子密度的分泌溶酶体向分泌口汇集时电子密度逐渐降低并降解为分泌小泡,其中的杆状结晶样毒蛋白也经无定型状态逐渐分散,经胞吐作用进入管腔并进一步疏散和均质化。免疫组化显示,分泌细胞颈部密集的分泌颗粒的主要成分为毒蛋白,毒蛋白在分泌细胞中呈明显的向心式梯度增强型分布。根据上述观察,提出了蜈蚣毒液以分泌溶酶体介导的非经典途径分泌的观点。     

FK506结合蛋白12.6调节小鼠嗜铬细胞分泌

FK506结合蛋白12.6(FKBP12.6)能够结合并调控钙离子释放通道兰尼碱受体2型(RyR2)的开放,可能是儿茶酚胺分泌的重要调控器.利用FKBP12.6敲除小鼠模型,我们研究了FKBP12.6在肾上腺嗜铬细胞胞吐中的作用.结果表明,FKBP12.6在小鼠肾上腺嗜铬细胞中表达,而敲除FKBP12.6小鼠的嗜铬细胞中有正常的去极化引起的钙电流和胞吐作用.然而,FKBP12.6敲除会导致嗜铬细胞中出现增强的咖啡因引起的细胞整体钙瞬变和咖啡因引起的胞吐作用.结果提示,FKBP12.6调控肾上腺嗜铬细胞儿茶酚胺的分泌,这种调控作用是通过调节钙离子的释放而实现的.FKBP12.6是嗜铬细胞分泌的重要蛋白.     

溶酶体离子通道蛋白在神经退行性疾病发生发展中的作用及机制研究

溶酶体离子通道蛋白异常引起溶酶体功能障碍是导致阿尔茨海默病(Alzheimer’s disease,AD)和帕金森病(Parkinson’s disease,PD)等神经退行性疾病的重要因素.溶酶体离子通道蛋白调节溶酶体内离子稳态、溶酶体膜电压以及溶酶体的酸度.溶酶体离子通道蛋白的结构或功能缺陷会引起溶酶体降解功能障碍,导致神经退行性疾病的发生发展.在这篇综述中,我们总结了各种离子通道蛋白调节溶酶体功能的过程及机制,以及离子通道蛋白异常参与神经退行性疾病的过程和机制.调节离子通道蛋白改善溶酶体的功能、促进异常聚集蛋白的清除,是神经退行性疾病治疗的潜在途径.     

丝状真菌胞吐体的研究进展

胞吐体(Exocyst)是真核生物细胞内由8个亚基组成的复合体,在后高尔基体产生的分泌囊泡与细胞膜特定位置的定向与拴系过程中发挥重要作用。一些小G蛋白通过直接与胞吐体的亚基相互作用,对极性胞外分泌进行精确的时空调节。丝状真菌的生长是通过菌丝顶端的极性生长来完成的,并且具有极强的分泌蛋白质能力,是研究胞吐体的理想系统。胞吐体对丝状真菌的形态发生和致病性都有极大的影响。本文综述了当前在丝状真菌中胞吐体的研究进展,包括它的组成、亚基定位、相关功能和调控。     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

本文利用激光扫描共聚焦显微镜A-CAS570从细胞形态学和功能两方面,研究了刀豆素A(Concanavalin A,Con A)、麦芽凝集素(Wheat Germ Agglutinin,WGA)、酵母多糖(Zymosan A,Z.A)对小鼠腹腔巨噬细胞胞质pH和溶酶体内荧光探针FITC—Dextran排出细胞的影响。结果显示三种配体加入细胞外液10min内,胞质pH很快下降,此后维持在该水平;在15min左右细胞外FITC一Dextran迅速增加,20min后变化趋于停止;在三种配体加入后15min左右,细胞内溶酶体在质膜内侧增多;25—30min溶酶体重新向细胞中央运动。根据上述实验结果,我们认为溶酶体pH升高是触发溶酶体内荧光探针通过胞吐作用排出细胞的必要条件,胞质酸化抑制溶酶体内容物通过胞吐作用排出细胞。配体刺激引起的溶酶体内容物通过胞吐作用排出细胞和胞质酸化是细胞自我调节和保护的一种反映。     

内分泌细胞中溶酶体对激素分泌调节的作用

本实验用外源性雄激素引起垂体促性腺激素细胞和睾丸间质细胞分泌抑制,对这两种细胞中的溶酶体及分泌吞噬和自体吞噬活动进行了超微结构形态观察和半定量分析。实验中应用了CMP酶细胞化学技术和免疫胶体金技术。研究结果显示,在分泌受抑制状态下,垂体促性腺激素细胞中溶酶体增多,分泌吞噬活动加强;与此同时,睾丸间质细胞也表现溶酶体增多、自体吞噬活动加强。这些结果不仅再次证明在分泌蛋白质激素细胞中溶酶体以分泌吞噬的方式参与了激素分泌调节,更重要的是初步证明在分泌类固醇激素细胞的分泌调节中,也有溶酶体的参与,其形式是自体吞噬作用。细胞通过自体吞噬作用得以在短时间内清除一部分合成激素的细胞器和其中的激素,这可能是分泌类固醇激素的细胞及时有效地调整激素分泌量的一项重要机制,与分泌蛋白质激素细胞的分泌吞噬有着相同的意义。     

Identification of SNAREs involved in synaptotagmin VII-regulated lysosomal exocytosis

Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.     

cAMP regulates Ca2+-dependent exocytosis of lysosomes and lysosome-mediated cell invasion by trypanosomes.

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis

Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.     

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.     

The small chemical vacuolin-1 alters the morphology of lysosomes without inhibiting Ca2+-regulated exocytosis

Ca2+-regulated exocytosis of lysosomes was previously shown to be required for the repair of plasma membrane wounds. The small chemical vacuolin-1 alters the morphology of lysosomes without affecting the ability of cells to reseal their plasma membrane after injury. On the basis of a failure to detect Ca2+-triggered lysosomal exocytosis in vacuolin-1-treated cells, a recent study proposed that lysosomes are dispensable for resealing. Here, we show that vacuolin-1, despite altering lysosome morphology, does not inhibit the exocytosis of lysosomes induced by exposure to a Ca2+ ionophore, or by plasma membrane wounding. Thus, lysosomes cannot be excluded as agents of membrane repair in vacuolin-1-treated cells.     

Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.     

Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes

Plasma membrane wounds are repaired by a mechanism involving Ca(2+)-regulated exocytosis. Elevation in intracellular [Ca(2+)] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca(2+)-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C(2)A domain or anti-Syt VII C(2)A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca(2+)-regulated exocytic compartments responsible for plasma membrane repair.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.