REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

FINE STRUCTURE OF THE SYNAPTIC DISCS SEPARATED FROM THE GOLDFISH MEDULLA OBLONGATA

Synaptic discs are structures localized in the club ending synapses on the Mauthner cell lateral dendrite of the goldfish medulla oblongata. The synaptic discs present a hexagonal array of particles ~8.5 nm center-to-center when observed in en face view. This lattice covers the entire surface Divalent cations are important in the stabilization of this particular hexagonal array of particles When a synaptic disc-rich fraction is treated with chelating agents (EDTA or EGTA), definite changes occur in the hexagonal lattice. First, the synaptic membranes show zones without particles interspersed with zones covered with the hexagonal array of particles Second, the synaptic discs break down and a new structure characterized by two parallel dense bands (7 nm each), separated by a 4 nm gap, is observed. The negative stain fills the gap region showing striations spaced ~10 nm center-to-center crossing the gap, but it does not penetrate the dense bands This "double band" structure is interpreted as an edge on view of a fragment of the synaptic membrane complex. Further treatment of this fraction with a chelating agent plus 0.3% deoxycholate produces an increase in the number of double band structures. However, EDTA plus Triton X-100 (a treatment known to produce solubilization of membrane proteins) never shows such double band structure An ordered material was observed associated with the cytoplasmic leaflets of the double bands This material consists of rows of beads ~4 nm in diameter and spaced at intervals of ~7 nm. Each of these beads is joined to the band by a thin stalk.     

Emerging roles of cortical microtubule–membrane interactions

Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions.     

The Oligomeric Outer Dynein Arm Assembly Factor CCDC103 Is Tightly Integrated within the Ciliary Axoneme and Exhibits Periodic Binding to Microtubules

CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other dynein or docking complex components for its integration. Unlike other known dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 m NaCl and requires more chaotropic conditions, such as 0.5 m KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm dynein repeat. Although the outer dynein arm docking complex is necessary to form arrays of dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice.     

Structural basis of membrane budding by the nuclear egress complex of herpesviruses

During nuclear egress, herpesvirus capsids bud at the inner nuclear membrane forming perinuclear viral particles that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytoplasm. This unusual budding process is mediated by the nuclear egress complex (NEC) composed of two conserved viral proteins, UL31 and UL34. Earlier, we discovered that the herpesvirus nuclear egress complex (NEC) could bud synthetic membranes in vitro without the help of other proteins by forming a coat‐like hexagonal scaffold inside the budding membrane. To understand the structural basis of NEC‐mediated membrane budding, we determined the crystal structures of the NEC from two herpesviruses. The hexagonal lattice observed in the NEC crystals recapitulates the honeycomb coats within the budded vesicles. Perturbation of the oligomeric interfaces through mutagenesis blocks budding in vitro confirming that NEC oligomerization into a honeycomb lattice drives budding. The structure represents the first atomic‐level view of an oligomeric array formed by a membrane‐deforming protein, making possible the dissection of its unique budding mechanism and the design of inhibitors to block it.     

FEEDING APPARATUS OF THE COLORLESS FLAGELLATE KATABLEPHARIS (CRYPTOPHYCEAE)1

The feeding apparatus of Kalablepharis ovalis (isolated from a freshwater impoundment in Colorado) and Katablepharis clone G-2 (isolated from the littoral of the Black Sea near Yalta in the Crimea) consists of inner and outer oval-shaped arrays of microtubules that begin at the anterior end of the cell and pass into the posterior of the cell. Each array of microtubules contains groups of microtubules with two to eight microtubules per group depending on the position of the array in the cell. A specialized area of the plasma membrane, the mouth, occurs at the anterior end of the cell. The mouth is oval with the long axis oriented dorsoventrally and consists of a raised ridge surrounding a central depression. The anterior end of the microtubules of the inner and outer arrays supports the raised ridge of the mouth. In freeze-fracture replicas, the protoplasmic face of the plasma membrane contains intramembrane particles on the raised ridge of the mouth. Three small membrane-cisternae occur on the protoplasmic side of the plasma membrane in the area of the mouth. Katablepharis clone G-2 also has five or six additional large membrane-cisternae associated with the inner microtubular array in the anterior portion of the cell. These larger membrane-cisternae do not occur in K. ovalis. Vesicles with electron-dense contents occur in association with the microtubular arrays. Katablepharis ovalis has a second type of vesicle containing a single-membrane profile associated with the microtubule arrays. The structure of the microtubular arrays in Katablepharis is compared with similar structures in suctorian ciliates and dinoflagellates.     

MEMBRANE MODIFICATIONS IN THE APICAL ENDOCYTIC COMPLEX OF ILEAL EPITHELIAL CELLS

Ileal lining cells of the suckling rat possess an "apical endocytic complex" capable of sequestering intact protein from the intestinal lumen. The complex consists of a network of invaginations of the apical plasma membrane, a number of subjacent small vesicles, and a giant supranuclear vacuole. The first two components initially incorporate material from the intestinal lumen and then transfer it to the giant vacuole where it is stored. Their limiting membrane displays striking structural modifications when viewed in various planes of section. Its lumenal dense leaflet appears discontinuous and consists of an ordered array of minute discrete plaques. A dense particle approximately 70 A in diameter is centered over each plaque. The particles are arranged in a two-dimensional square lattice with center-to-center spacing of approximately 120 A.     

Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder.

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.     

Morphology and molecular composition of isolated postsynaptic junctional structures.

The solubilization of isolated brain synaptosomal plasma membranes by various detergents was studied and in each case found to depend upon detergent concentration. By using conditions sufficient to extract maximally protein and phospholipid from the membranes, postsynaptic junctional particles were isolated with each of four detergents and their ultrastructural appearances and protein contents compared. Two basic structural forms were identified. One, isolated with Triton X-100, consists of a planar array of dense-staining particles ca. 20 nm in diameter. It resembles the postsynaptic density seen in undigested synaptosomal plasma membranes. The other, isolated with sodium deoxycholate, contains less protein. It has the same overall shape and dimensions as the postsynaptic density, but consists of a branching network of short 5 nm fibres (the postsynaptic junctional lattice) making up an array of contiguous polygons, each ca. 20 nm across. The interior of these polygonal elements seems to be hydrophobic since it cannot be penetrated by metallic salts used for negative staining. It is suggested that the dense-staining 20 nm subunits observed at the postsynaptic junctional site may be composed of hydrophobic proteins inserted into the hollow cores of the lattice polygons. Electrophoretic analysis of the proteins present in the various postsynaptic junctional preparations identified two major common components with molecular masses of 275000 and 47500. The latter is tentatively identified as actin. Components comigrating respectively with alpha- and beta-tubulin are present, and the relation of the lattice structure to subjacent microtubules is discussed.     

Making parallel lines meet: Transferring information from microtubules to extracellular matrix

The extracellular matrix is constructed beyond the plasma membrane, challenging mechanisms for its control by the cell. In plants, the cell wall is highly ordered, with cellulose microfibrils aligned coherently over a scale spanning hundreds of cells. To a considerable extent, deploying aligned microfibrils determines mechanical properties of the cell wall, including strength and compliance. Cellulose microfibrils have long been seen to be aligned in parallel with an array of microtubules in the cell cortex. How do these cortical microtubules affect the cellulose synthase complex? This question has stood for as many years as the parallelism between the elements has been observed, but now an answer is emerging. Here, we review recent work establishing that the link between microtubules and microfibrils is mediated by a protein named cellulose synthase-interacting protein 1 (CSI1). The protein binds both microtubules and components of the cellulose synthase complex. In the absence of CSI1, microfibrils are synthesized but their alignment becomes uncoupled from the microtubules, an effect that is phenocopied in the wild type by depolymerizing the microtubules. The characterization of CSI1 significantly enhances knowledge of how cellulose is aligned, a process that serves as a paradigmatic example of how cells dictate the construction of their extracellular environment.     

Freeze fracture study of Toxoplasma and Sarcocystis infective stages (author's transl)]

Infective stages of Toxoplasma and Sarcocystis have been studied by the freeze fracture technique. The outer membrane of the pellicle is continuous and shows an apical 8 + 1 particles rosette in the P fracture face. The inner membrane complex is made of rectangular flattened vesicles aligned in longitudinal rows and joined in a puzzle like fashion. Sarcocystis has 11 of these rows whereas their number varies in Toxoplasma. A posterior interruption is present. Anteriorly is one truncated conical plate apically opened by a vertical ridge. The membranes of the inner complex are characterized by parallel alignment of particles (in P faces) some of which are joined and continuous with double rows radiating in the apical cap. Those rows correspond in number and arrangement with the underlying microtubules. The rhoptries membranes show periodic circular arrays of particles.     

How cellular membranes can regulate microtubule network

Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing “tracks” for their subsequent transport.     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

Higher plant cortical microtubule array analyzed in vitro in the presence of the cell wall

Plant morphogenesis depends on an array of microtubules in the cell cortex, the cortical array. Although the cortical array is known to be essential for morphogenesis, it is not known how the array becomes organized or how it functions mechanistically. Here, we report the development of an in vitro model that provides good access to the cortical array while preserving the array's organization and, importantly, its association with the cell wall. Primary roots of maize (Zea mays) are sectioned, without fixation, in a drop of buffer and then incubated as desired before eventual fixation. Sectioning removes cytoplasm except for a residuum comprising cortical microtubules, vesicles, and fragments of plasma membrane underlying the microtubules. The majority of the cortical microtubules remain in the cut-open cells for more than 1 h, fully accessible to the incubation solution. The growth zone or more mature tissue can be sectioned, providing access to cortical arrays that are oriented either transversely or obliquely to the long axis of the root. Using this assay, we report, first, that cortical microtubule stability is regulated by protein phosphorylation; second, that cortical microtubule stability is a function of orientation, with divergent microtubules within the array depolymerizing within minutes of sectioning; and third, that the polarity of microtubules in the cortical array is not uniform. These results suggest that the organization of the cortical array involves random nucleation followed by selective stabilization of microtubules formed at the appropriate orientation, and that the signal specifying alignment must treat orientations of +/- 180 degrees as equivalent.     

Disassembly and reconstitution of a membrane-microtubule complex

The cell membrane of the unicellular algae Distigma proteus is associated with arrays of parallel microtubules. Fragments of the membrane-microtubule complex have been isolated and partially purified. The microtubules were stable in vitro at room temperature as well as at 0 degree C, but were specifically and rapidly disassembled by Ca2+. After removal of all endogenous microtubules, the membrane-microtubule complex could be reassembled from brain microtubule protein and denuded Distigma membrane fragments. The readded microtubules bound in a fixed orientation, and only to those regions of membrane that are normally associated with microtubules in vivo.     

The parallel lives of microtubules and cellulose microfibrils

A major breakthrough was the recent discovery that cellulose synthases really do move along the plasma membrane upon tracks provided by the underlying cortical microtubules. It emphasized the cytoplasmic contribution to cell wall organization. A growing number of microtubule-associated proteins has been identified and shown to affect the way that microtubules are ordered, with downstream effects on the pattern of growth. The dynamic properties of microtubules turn out to be key in understanding the behaviour of the global array and good progress has been made in deciphering the rules by which the array is self-organized.     

Cryofracture electron microscopy of the ookinete pellicle of Plasmodium gallinaceum reveals the existence of novel pores in the alveolar membranes

The malaria parasite invades the midgut tissue of its mosquito host as a motile form called the ookinete. We have examined the pellicle of the ookinete of Plasmodium gallinaceum by freeze-fracture and quick-freeze, deep-etch electron microscopy. The general organization is analogous to that of invasive stages of other members of Apicomplexa. The pellicle is composed of three membranes: the plasma membrane, and the two linked intermediate and inner membranes, which in the ookinete form one flattened vacuole that is located beneath the plasma membrane. The edges of this vacuole form a longitudinal suture. Beneath the vacuole is found an array of microtubules that are connected to the inner membrane by intramembranous particles. During freeze-fracture, the membranes can split along their hydrophobic planes, thus yielding six fracture faces, each of which displays a characteristic pattern of intramembranous particles. Additionally, we find that the ookinete pellicle differs from all other apicomplexan motile stages by the presence of large pores. These pores are of unknown function, but clearly might constitute a novel pathway for the transport of molecules to and from the cortex, which is independent of the well-described route through the apical micronemal/rhoptry complex. The pores may be the route by which motor proteins or other non micronemal surface proteins are trafficked, such as P25/P28 and SOAP, some of which are implicated in transmission blocking immunity.     

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.     

The Trypanosoma brucei cytoskeleton: Ultrastructure and localization of microtubule-associated and spectrin-like proteins using quick-freeze, deep-etch, immunogold electron microscopy

We have used a combination of quick-freezing/deep-etching and colloidal gold immunocytochemistry (i) to analyze the molecular organization of the microtubular membrane skeleton and the flagellum of Trypanosoma brucei, and (ii) to localize two defined cytoskeletal proteins within these structures. The cell body of trypanosomatids is enveloped by a membrane skeleton consisting of a tightly packed array of microtubules which are closely associated with the cell membrane. The membrane-oriented face of these microtubules is richly decorated with microtubule-associated proteins, which form intermicrotubule and microtubule-membrane linkers. In contrast, the cytoplasmic faces of the microtubules have a smooth, nondecorated appearance. A previously identified, highly repetitive microtubule-associated protein is confined to the membrane-oriented face of the microtubular array, suggesting that the function of this protein may be that of a microtubule-membrane linker. Quickfreezing has also been used to reveal the geometric organization of the paraflagellar rod structure in the flagellum, its interaction with the cell body, and a unique series of fleur-de-lis-like molecules which link this organelle to axonemal microtubules. Immunohistochemistry using an antibody against human erythrocyte spectrin suggests that these linker structures may contain ancestral spectrin-like molecules.