Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism

ABSTRACT: BACKGROUND: Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. RESULTS: Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. CONCLUSION: Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.     

Vesicle trafficking and membrane remodelling in cytokinesis

All cells complete cell division by the process of cytokinesis. At the end of mitosis, eukaryotic cells accurately mark the site of division between the replicated genetic material and assemble a contractile ring comprised of myosin II, actin filaments and other proteins, which is attached to the plasma membrane. The myosin-actin interaction drives constriction of the contractile ring, forming a cleavage furrow (the so-called 'purse-string' model of cytokinesis). After furrowing is completed, the cells remain attached by a thin cytoplasmic bridge, filled with two anti-parallel arrays of microtubules with their plus-ends interdigitating in the midbody region. The cell then assembles the abscission machinery required for cleavage of the intercellular bridge, and so forms two genetically identical daughter cells. We now know much of the molecular detail of cytokinesis, including a list of potential genes/proteins involved, analysis of the function of some of these proteins, and the temporal order of their arrival at the cleavage site. Such studies reveal that membrane trafficking and/or remodelling appears to play crucial roles in both furrowing and abscission. In the present review, we assess studies of vesicular trafficking during cytokinesis, discuss the role of the lipid components of the plasma membrane and endosomes and their role in cytokinesis, and describe some novel molecules implicated in cytokinesis. The present review covers experiments performed mainly on tissue culture cells. We will end by considering how this mechanistic insight may be related to cytokinesis in other systems, and how other forms of cytokinesis may utilize similar aspects of the same machinery.     

‘Life is a Highway’: Membrane Trafficking During Cytokinesis

Cytokinesis, the final stage of the cell cycle, is an essential step toward the formation of two viable daughter cells. In recent years, membrane trafficking has been shown to be important for the completion of cytokinesis. Vesicles originating from both the endocytic and secretory pathways are known to be shuttled to the plasma membrane of the ingressing cleavage furrow, delivering membrane and proteins to this dynamic region. Advances in cell imaging have led to exciting new discoveries regarding vesicle movement in living cells. Recent work has revealed a significant role for membrane trafficking, as controlled by regulatory proteins, during cytokinesis in animal cells. The endocytic and secretory pathways as well as motor proteins are revealed to be essential in the delivery of vesicles to the cleavage furrow during cytokinesis.     

Molecular mechanisms of contractile-ring constriction and membrane trafficking in cytokinesis

In this review, we discuss the molecular mechanisms of cytokinesis from plants to humans, with a focus on contribution of membrane trafficking to cytokinesis. Selection of the division site in fungi, metazoans, and plants is reviewed, as well as the assembly and constriction of a contractile ring in fungi and metazoans. We also provide an introduction to exocytosis and endocytosis, and discuss how they contribute to successful cytokinesis in eukaryotic cells. The conservation in the coordination of membrane deposition and cytoskeleton during cytokinesis in fungi, metazoans, and plants is highlighted.     

Endocytosis resumes during late mitosis and is required for cytokinesis

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.     

Members of the Arabidopsis dynamin-like gene family,ADL1, are essential for plant cytokinesis and polarized cell growth

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.     

A rich and bountiful harvest: Key discoveries in plant cell biology

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke’s discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Seven senior plant cell biologists reflect on foundational contributions to a variety of topics, including pollen tube signaling, cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology.     

High lipid order of Arabidopsis cell‐plate membranes mediated by sterol and DYNAMIN‐RELATED PROTEIN1A function

Membranes of eukaryotic cells contain high lipid‐order sterol‐rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher‐plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell‐plate formation in Arabidopsis relies on sterol‐ and DYNAMIN‐RELATED PROTEIN1A (DRP1A)‐dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order‐sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid‐order membrane domain. The cell‐plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co‐localize with DRP1A at the cell plate, and DRP1A accumulates in detergent‐resistant membrane fractions. Modifications of sterol concentration or composition reduce cell‐plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid‐order domain, and pave the way to explore potential feedback between lipid order and function of dynamin‐related proteins during cytokinesis.     

Roles of septins in the mammalian cytokinesis machinery

Septins comprise a eukaryotic guanine nucleotide binding protein subfamily which form filamentous heteropolymer complexes. Although mechanism of cytokinesis is diverged by species and tissues, loss of septin function results in the multinuclear phenotype in many organisms. Hence septin filaments beneath the cleavage furrow are hypothesized as a structural basis to ensure completion of cytokinesis. However, molecular mechanisms of septin assembly, disassembly and function have been elusive despite the potential importance of this ubiquitous cytoskeletal system. Meanwhile, growing evidence suggests that mammalian septins functionally or physically interact with diverse molecules such as actin, actin-binding proteins, proteins of membrane fusion machinery, Cdc42 adapter proteins, a ubiquitin-protein ligase, and phosphoinositides. Careful integration of these data may provide insights into the mechanism of mammalian septin organization and functions in cytokinesis.     

Cytokinesis in plant and animal cells: endosomes 'shut the door'

For many years, cytokinesis in eukaryotic cells was considered to be a process that took a variety of forms. This is rather surprising in the face of an apparently conservative mitosis. Animal cytokinesis was described as a process based on an actomyosin-based contractile ring, assembling, and acting at the cell periphery. In contrast, cytokinesis of plant cells was viewed as the centrifugal generation of a new cell wall by fusion of Golgi apparatus-derived vesicles. However, recent advances in animal and plant cell biology have revealed that many features formerly considered as plant-specific are, in fact, valid also for cytokinetic animal cells. For example, vesicular trafficking has turned out to be important not only for plant but also for animal cytokinesis. Moreover, the terminal phase of animal cytokinesis based on midbody microtubule activity resembles plant cytokinesis in that interdigitating microtubules play a decisive role in the recruitment of cytokinetic vesicles and directing them towards the cytokinetic spaces which need to be plugged by fusing endosomes. Presently, we are approaching another turning point which brings cytokinesis in plant and animal cells even closer. As an unexpected twist, new studies reveal that both plant and animal cytokinesis is driven not so much by Golgi-derived vesicles but rather by homotypically and heterotypically fusing endosomes. These are generated from cytokinetic cortical sites defined by preprophase microtubules and contractile actomyosin ring, which induce local endocytosis of both the plasma membrane and cell wall material. Finally, plant and animal cytokinesis meet together at the physical separation of daughter cells despite obvious differences in their preparatory events.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Regulation of cytokinesis by membrane trafficking involving small GTPases and the ESCRT machinery

During cell division, cells undergo membrane remodeling to achieve changes in their size and shape. In addition, cell division entails local delivery and retrieval of membranes and specific proteins as well as remodeling of cytoskeletons, in particular, upon cytokinetic abscission. Accumulating lines of evidence highlight that endocytic membrane removal from and subsequent membrane delivery to the plasma membrane are crucial for the changes in cell size and shape, and that trafficking of vesicles carrying specific proteins to the abscission site participate in local remodeling of membranes and cytoskeletons. Furthermore, the endosomal sorting complex required for transport (ESCRT) machinery has been shown to play crucial roles in cytokinetic abscission. Here, the author briefly overviews membrane-trafficking events early in cell division, and subsequently focus on regulation and functional significance of membrane trafficking involving Rab11 and Arf6 small GTPases in late cytokinesis phases and assembly of the ESCRT machinery in cytokinetic abscission.     

Dynamin and cytokinesis

Animal and plant cytokineses appear morphologically distinct. Recent studies, however, have revealed that these cellular processes have many things in common, including the requirement of co-ordinated membrane trafficking and cytoskeletal dynamics. At the intersection of these two processes are the members of the dynamin family of ubiquitous eukaryotic GTPases. In this review, we highlight the conserved contribution of classical dynamin and dynamin-related proteins during cytokinesis in both animal and plant systems.     

RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis

In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.     

Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration

Integrin trafficking from and to the plasma membrane controls many aspects of cell behavior including cell motility, invasion, and cytokinesis. Recruitment of integrin cargo to the endocytic machinery is regulated by the small GTPase Rab21, but the detailed molecular mechanisms underlying integrin cargo recruitment are yet unknown. Here we identify an important role for p120RasGAP (RASA1) in the recycling of endocytosed α/β1-integrin heterodimers to the plasma membrane. Silencing of p120RasGAP attenuated integrin recycling and augmented cell motility. Mechanistically, p120RasGAP interacted with the cytoplasmic domain of integrin α-subunits via its GAP domain and competed with Rab21 for binding to endocytosed integrins. This in turn facilitated exit of the integrin from Rab21- and EEA1-positive endosomes to drive recycling. Our results assign an unexpected role for p120RasGAP in the regulation of integrin traffic in cancer cells and reveal a new concept of competitive binding of Rab GTPases and GAP proteins to receptors as a regulatory mechanism in trafficking.     

Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis

Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside G(M1) and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergent-resistant membranes contain Src and PLCgamma, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to cytokinesis.     

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Identification of Cdk targets that control cytokinesis

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.     

磷脂酰肌醇转移蛋白

磷脂酰肌醇转移蛋白(phosphatidylinositol/phosphatidylcholine transfer proteins,PITP)普遍存在于真核生物细胞中,PITP能够结合并交换一分子的磷脂酰肌醇(phosphatidylinositol,PI)或磷脂酰胆碱(phosphatidylcholine,PC),并促进这两类脂分子在细胞内膜组分间的转移。PITP对细胞内膜组分间脂类的运输和代谢、分泌囊泡的形成和运输、磷脂酶C(phospholipase,PLC)调节的信号传导以及神经退化等生理生化过程具有重要的影响。综述了近年来PITP的研究进展,并对目前研究中存在的一些问题进行探讨。