Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells

Although bulk endocytosis has been found in a number of neuronal and endocrine cells, the molecular mechanism and physiological function of bulk endocytosis remain elusive. In pancreatic beta cells, we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization. Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca²⁺ entry and suppressed by the inhibition of dynamin function. Moreover, defects in bulklike endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice, which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability.     

The mammalian endocytic cytoskeleton

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and invaginates to form a vesicle that buds off—along with its contents—into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.     

Endocytosis in plants: fact or artefact?

Whilst plant cells are apparently equipped with all the necessary molecular machinery for receptor-mediated endocytosis, the physiological role of this process in these cells remains an enigma. In this article, we consider current opinions of endocytosis in plants and define some of the problems that have impeded progress in our under-standing of the part played by endocytosis in the vesicle trafficking pathway.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Clathrin‐Mediated Endocytosis at the Synaptic Terminal: Bridging the Gap Between Physiology and Molecules

It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non‐neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here, we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Organisation and results of the cervical cytology screening programme in British Columbia, 1955-85

A screening programme to detect preinvasive carcinoma of the cervix was started in British Columbia in 1949. Since 1970 the number of women who have been screened at least once has been maintained at about 85% of the population at risk. More than 500 000 cervical smears are being examined each year in the central laboratory. There has been an appreciable increase in the number of cases and rates of carcinoma in situ seen since 1970, particularly in women between 20 and 30 years of age. Since the programme started over 26 000 cases of squamous carcinoma in situ have been detected and treated. The incidence of clinically invasive squamous carcinoma of the cervix has fallen by 78% during the period under review, and mortality from squamous carcinoma of the cervix has fallen by 72%. A colposcopy programme, introduced throughout British Columbia over the past 12 years, has been important in reducing the problems of managing preinvasive lesions, particularly in younger women.It is concluded that the reduction in morbidity and mortality from invasive squamous cancer of the cervix in British Columbia over the past 30 years is directly attributable to the province wide screening programme and that a large potential increase in invasive cervical cancer rates among younger women is being prevented.     

N-BAR and F-BAR proteins—endophilin-A3 and PSTPIP1—control clathrin-independent endocytosis of L1CAM

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.     

Endocytosis in plants

Endocytosis in animal cells has been heavily documented. Both fluid-phase and receptor-mediated modes of uptake have been frequently studied, and the endocytic pathway is well defined. This contrasts markedly with the situation in plants where our knowledge of this process is still rudimentary. Partly responsible for this situation has been the view, widely held among plant physiologists, that because of turgor, endocytosis cannot occur in plant cells. As discussed below, the case against endocytosis is no longer water-tight.
Endocytosis is a fact in protoplasts. It can be demonstrated with electron-dense tracers as well as with membrane impermeant dye Lucifer Yellow CH. The latter has also been used with success on both suspension-cultured and tissue cells of higher plants, suggesting that fluid-phase endocytosis is also a feature of cells with walls. Through the application of fluorescently labelled elicitor molecules, which specifically bind to the cell surface of suspension-cultured cells, it has also been possible to provide convincing evidence for the operation of receptor-mediated endocytosis in plants. A number of studies on protoplasts and cells clearly indicate that endocytosis in plants can be mediated by coated pits in the plasma membrane. At least one of the organelles that lie on the endocytic pathway in plants has a structurally similar counterpart in animal cells: the multivesicular body. The first recipient of internalized molecules is the partially coated reticulum, although its relationship to the Golgi apparatus and Golgi function remain to be clarified. The final target for endocytosis in plants appears to be the vacuole.     

Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON‐REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.     

Value of models for membrane budding

The budding of membranes and curvature generation is common to many forms of trafficking in cells. Clathrin-mediated endocytosis, as a prototypical example of trafficking, has been studied in great detail using a variety of experimental systems and methods. Recently, advances in experimental methods have led to great strides in insights on the molecular mechanisms and the spatiotemporal dynamics of the protein machinery associated with membrane curvature generation. These advances have been ably supported by computational models, which have given us insights into the underlying mechanical principles of clathrin-mediated endocytosis. On the other hand, targeted experimental perturbation of membranes has lagged behind that of proteins in cells. In this area, modeling is especially critical to interpret experimental measurements in a mechanistic context. Here, we discuss the contributions made by these models to our understanding of endocytosis and identify opportunities to strengthen the connections between models and experiments.     

Novel functions of endocytic player clathrin in mitosis

Clathrin has been widely recognized as a pivotal player in endocytosis, in which several adaptors and accessory proteins are involved. Recent studies suggested that clathrin is also essential for cell division. Here this review mainly focuses on the clathrin-dependent mechanisms involved in spindle assembly and chromosome alignment. In mitosis, clathrin forms a complex with phosphorylated TACC3 to ensure spindle stability and proper chromosome alignment. The clathrin-regulated mechanism in mitosis requires the crosstalk among clathrin, spindle assembly factors (SAFs), Ran-GTP and mitotic kinases. Meanwhile, a coordinated mechanism is required for role transitions of clathrin during endocytosis and mitosis. Taken together, the findings of the multiple functions of clathrin besides endocytosis have expanded our understanding of the basic cellular activities.     

Establishment and optimization of epithelial cell cultures from human ectocervix,transformation zone,and endocervix optimization of epithelial cell cultures

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.     

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.     

Nuclear Trafficking and Functions of Endocytic Proteins Implicated in Oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin‐mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.     

HLA types in women with cervical human papillomavirus (HPV) lesions prospectively followed up for 10 years

Certain genotypes of HPV have been recently implicated in the etiology of carcinoma of the uterine cervix. In order to determine whether HLA antigen‐controlled immunoregulatory functions have a role in the pathogenesis of HPV infections, class I and II HLA antigen typing was carried out on a series of 96 randomly selected women who were part of a cohort of 530 women prospectively followed up for cervical HPV infections in our clinic since 1981. The frequency of the DQ3 antigen, which has previously been reported to be increased among cervical cancer patients, was decreased in our HPV patients compared with the control group of Finnish women, but it was slightly increased in HPV16‐infected women ( P =0.0812). However, we were able to demonstrate that HLA‐DR5 antigen is significantly increased (i) in patients with high grade cervical intraepithelial neoplasia (CIN) ( P <0.02), and (ii) in women harbouring the high risk HPV type 16 ( P =0.0003), thus confirming earlier reports of an association of this HLA antigen and cervical cancer. Such a close association between the high risk HPV type 16 with an HLA antigen might have important implications in the possible immunogenetic basis of the increased risk for squamous cell carcinoma of the uterine cervix.     

Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin‐mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin‐coated pits and bulk endocytosis‐like structures increase on the plasma membrane in Syt11‐knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.     

植物胞吞和胞吐的耦合调控

真核细胞通过胞吞和胞吐作用将大分子和颗粒性物质运出或运送至质膜,其中包括一些具有重要生物学功能的蛋白质。胞吞和胞吐途径之间的耦合对维持质膜的完整性以及调控质膜蛋白的丰度和活性至关重要。动物中,突触小泡的胞吞和胞吐在时空上紧密耦合已被证明是持续神经传递的必要条件。近年来,随着对植物囊泡运输的深入研究,越来越多的证据表明,植物细胞的胞吞和胞吐间同样存在耦合调控,且在植物生长发育和对外界环境的响应中扮演重要角色。该文综述了植物协同调控胞吞和胞吐的生理学意义,并结合网格蛋白介导囊泡运输的最新研究进展探讨了其可能的耦合机制。     

PDGF—D和VEGF在宫颈鳞癌中的表达及其意义

目的：检测宫颈鳞癌组织中血小板源性生长因子D（PDGF—D）和血管内皮生长因子（VEGF）的表达,探讨二者在宫颈癌变过程中的作用及意义,为探讨宫颈鳞癌的发病机制和宫颈鳞癌的早期治疗提供理论依据。方法：采用免疫组织化学（sP法）检测40例宫颈鳞癌和10例正常宫颈组织中PDGF—D和VEGF蛋白的表达,分析两者之间的相关性及其与临床病理特征之间的关系。结果：宫颈鳞癌组织中PDGF—D和VEGF蛋白的表达显著高于正常宫颈组织（P〈0．05）;PDGF—D和VEGF的表达与宫颈鳞癌的分化程度及淋巴结转移有关（P〈0．05）;与年龄及临床分期无关（P〉0．05）。Spearman相关分析发现PDGF—D与VEGF表达程度呈正相关（r=0．346,P〈0．05）。结论：1．PDGF—D和VEGF在宫颈鳞癌组织中特异性高表达,可能在宫颈鳞癌的发生、发展与转移中起着重要作用。2．PDGF-D和VEGF表达与宫颈鳞癌的分化程度及淋巴结转移有关,与年龄及临床分期无关,提示它们可能在宫颈鳞癌的浸润和转移及预后方面有重要的监测意义。3．PDGF-D和VEGF在宫颈鳞癌组织中的表达呈正相关,提示两者起着相互促进的作用,对PDGF—D和VEGF的联合检查,为临床实际应用提供了参考。