Vesicle Invagination Research Articles

Bilateral congenital cystic eye with intraocular tissue differentiation in a horse.

Congenital cystic eye (CCE) is a rare congenital ocular disease, which has been suggested to be caused by problems with optic vesicle invagination. This is characterized by the formation of intraorbital cystic lesions lined by undifferentiated neuroepithelium. However, cases involving ocular structures with varying degrees of differentiation have also been reported as CCE in humans and animals. In horses, CCE have only been reported as a case presenting as neuroepithelial-lined cysts without the formation of ocular structures. This paper describes large bilateral intraorbital cysts in a 1-day-old horse. The histopathological findings revealed that the cysts were covered by neuroepithelium with formation of the lens, ciliary body, and optic nerve, and it was diagnosed as CCE. This report provides further variations of CCE in horses.

The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex.

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent AnnexinV probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

Peptide-Lipid Interaction Sites Affect Vesicles’ Responses to Antimicrobial Peptides

This article presents coarse-grained molecular dynamics simulations of pore-forming antimicrobial peptide melittin and its interactions with vesicles composed of a mixture of zwitterionic and anionic phospholipids. Besides creating holes in the membrane, the adsorption of melittin also induces vesicle budding, which can develop into vesiculation at high peptide concentrations, as well as vesicle invagination, which can eventually result in a corrugated membrane surface. These rich morphology changes are mediated by the curvature of the vesicles and the peptide concentration. Highly curved vesicles favor the recruitment of melittins with a higher density of binding sites. The peptides mainly penetrate into the membrane surface in monomers via hydrophobic interaction. Lowly curved vesicles recruit melittins with a low density of binding sites. Surplus peptides are prone to form oligomers and shallowly adsorb on the surface of membrane via electrostatic interaction. The penetration of monomers induces membrane pore formation and positive membrane curvature, which promote vesicle budding. The adsorption of oligomers induces negative membrane curvature, which promotes vesicle invagination. This work demonstrates that antimicrobial peptides adopt multiple actions to destroy bacterial membranes.

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.

Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

From invagination to navigation: The story of magnetosome-associated proteins in magnetotactic bacteria.

Magnetotactic bacteria (MTB) are a group of Gram-negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome-chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome-associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme.

Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.

Morphogenetic Model for Radial Streaking in the Fundus of the Carrier State of X-Linked Albinism

Morphogenetic Model for Radial Streaking in the Fundus of the Carrier State of X-Linked Albinism

Sperm morphology of two species of Olivancillaria (Gastropoda: Olividae) from the south-western Atlantic

Sperm ultrastructure in two species of the marine snail family Olividae is examined. Euspermatozoa of both species are composed of a conical, membrane-bound acrosomal vesicle; an axial rod and a basal plate similar in both species; a solid and highly electron-dense nucleus; an elongate midpiece consisting of the axoneme sheathed by helical mitochondrial elements; an elongate glycogen piece; a double electron-dense ring at the junction of the midpiece and glycogen piece; and a free tail region. The slight narrowing in the acrosomal vesicle invagination is situated in different levels between Olivancillaria deshayesiana and Olivancillaria carcellesi. This morphology could be considered as a specific character. The length of the nucleus in O. carcellesi and in O. deshayesiana is shorter than that of other neogastropods, and could be diagnostic at family level.

The Arp2/3 activator WASH regulates α5β1-integrin-mediated invasive migration.

The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5β1 integrin recycling. WASH regulates the trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

Yeast dynamin implicated in endocytic scission and the disassembly of endocytic components

The yeast dynamin-related GTPase Vps1 has been implicated in a range of cellular functions including vacuolar protein sorting, protein trafficking, organization of peroxisome, and endocytosis.1-2 Vps1 is present at endocytic sites and may be directly involved in endocytic vesicle invagination through its membrane-tubulating activity. Here, evidence supporting the functional link between Vps1 and the yeast amphiphysin Rvs167 in vesicle invagination is discussed. Though the disassembly of endocytic factors from pinched-off endocytic vesicles appears to be tightly regulated in a spatiotemporal manner, we are far from having complete understanding of the underlying mechanism. In this study, we provide evidence that Vps1 plays a role in the uncoating of endocytic proteins from post-internalized vesicles, based on the observation of a quick disassembly of two endocytic coat proteins Ent1 and Ent2 in cells lacking Vps1.

Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients.

The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal α-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system

It is known that two cytoskeleton components, microtubules and actins filaments, are required for efficient endocytosis. The relative importance of these two components in the cellular uptake of 2-(dimethylamino)ethyl methacrylate (DMAEMA)-DNA polyplexes was investigated in this study by applying microtubule depolymerising agent, colchicine, and actin polymerising inhibitor, cytochalasin D, in a cell transfection study. The effect of colchicine on transfection efficiency of polyplexes was found to be a time-dependent phenomenon, whereby the level of gene expression was inhibited at early stage, presumably to the disruption of a transport of vesicles along microtubules by colchicine. As time progressed, the level of gene expression was significantly enhanced relative to the control, possibly due to the failure in transport of vesicles from endosomes to late lysosomes, or due to the breakdown of nuclear membrane when mitosis was arrested at metaphase by colchicine. On the other hand, transfection efficiency was significantly reduced at all time points by cytochalasin D, which is considered to primarily affects invagination of vesicles at the early stage of endocytosis by inhibiting actin polymerisation. Further investigation to identify the endocytotic route of DMAEMA polyplexes was conducted applying clathrin- and caveolae- pathways inhibitors in cell transfection study. The results indicate that DMAEMA polyplexes were internalized primarily through clathrin-mediated pathway, with a minor fraction possibly entering cells via a caveolae-mediated pathway.

Amphiphysin I and regulation of synaptic vesicle endocytosis.

Amphiphysin I, known as a major dynamin-binding partner localized on the collars of nascent vesicles, plays a key role in clathrin-mediated endocytosis (CME) of synaptic vesicles. Amphiphysin I mediates the invagination and fission steps of synaptic vesicles by sensing or facilitating membrane curvature and stimulating the GTPase activity of dynamin. Amphiphysin I may form a homodimer by itself or a heterodimer with amphiphysin II in vivo. Both amphiphysin I and II function as multilinker proteins in the clathrin-coated complex. Under normal physiological conditions, the functions of amphiphysin I and some other endocytic proteins are known to be regulated by phosphorylation and dephosphorylation. During hyperexcited conditions, the most recent data showed that amphiphysin I is truncated by the ca2-dependent protease calpain. Overexpression of the truncated form of amphiphysin I inhibited transferrin uptake and synaptic vesicle endocytosis (SVE). This suggests that amphiphysin I may be an important regulator for SVE when massive amounts of Ca2 flow into presynaptic terminals, a phenomenon observed in neurodegenerative disorders such as ischemia/anoxia, epilepsy, stroke, trauma and Alzheimer's disease. This review describes current knowledge regarding the general properties and functions of amphiphysin I as well as the functional regulations such as phosphorylation and proteolysis in nerve terminals.

Loss of Tbx2 delays optic vesicle invagination leading to small optic cups

Tbx2 is a T-box transcription factor gene that is dynamically expressed in the presumptive retina during optic vesicle invagination. Several findings implicate Tbx2 in cell cycle regulation, including its overexpression in tumours and regulation of proliferation during heart development. We investigated the role of Tbx2 in optic cup formation by analysing mice with a targeted homozygous mutation in Tbx2. Loss of Tbx2 caused a reduced presumptive retinal volume due to increased apoptosis, and a delay in ventral optic vesicle invagination leading to the formation of small and abnormally shaped optic cups. Tbx2 is essential for maintenance, but not induction of expression of the dorsal retinal determinant, Tbx5, and acts downstream of Bmp4, a dorsally expressed gene implicated in human microphthalmia. The small retina showed a hypocellular ventral region, loss of Fgf15, normally expressed in proliferating central retinal cells, and increased numbers of mitotic cells in the dorsal region, indicating that Tbx2 is required for normal growth and development across the D-V axis. Dorsal expression of potential regulators of retinal growth, Cyp1b1 and Cx43, and the topographic guidance molecule ephrinB2, was increased, and intraretinal axons were disorganised resulting in a failure of optic nerve formation. Our data provide evidence that Tbx2 is required for proper optic cup formation and plays a critical early role in regulating regional retinal growth and the acquisition of shape during optic vesicle invagination.

Membrane scission by the ESCRT-III complex

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.

Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

BackgroundHTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins.ResultsHere, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding.ConclusionThese findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus.

Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation

Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca(2+)-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca(2+) influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.

There goes the neighborhood. Focus on “Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion”

the membranes of eukaryotic cells contain small amounts of phosphoinositides, which are phosphorylated forms of the membrane lipid phosphatidylinositol (PtdIns). Together, PtdIns and its phosphoinositide derivatives constitute ∼10% of the total cellular lipid in most cells. Although