Microtubules in Transcellular Movement of Procollagen

Alteration of microtubular funtion modifies the rate at which procollagen is converted to collagen.     

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules

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Intracellular Transformation of Phagosomes

The transformation of nascent phagosomes into forms capable of interacting with antimicrobial organelles of phagocytes, peroxisomes, depends on certain interactions between phagosomes and other vacuolar organelles. Phagosomes repeatedly interact with early and late endosomes through temporary contacts, which allows them to gain and lose complex sets of proteins. In addition, certain polypeptides are eliminated from phagosomes through recycling. New proteins enter phagosomes from the organelles of the biosynthetic pathway or are recruited from the cytoplasm. In addition, phagosomes receive proteins in the process of interaction with endosomes. The overall result of such transformation is acquiring new properties that make possible their interaction with peroxisomes.     

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.     

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.     

Modelling the Role of Intrinsic Electric Fields in Microtubules as an Additional Control Mechanism of Bi-directional Intracellular Transport

Active transport is essential for cellular function, while impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is clear that many types of cargo are moved by both sets of motors, and frequently in a reverse direction. The general question of how the direction of transport is regulated is still open. The mechanism of the cell's differential control of diverse cargos within the same cytoplasmic background is still unclear as is the answer to the question how endosomes and mitochondria move to different locations within the same cell. To answer these questions we postulate the existence of a local signaling mechanism used by the cell to specifically control different cargos. In particular, we propose an additional physical mechanism that works through the use of constant and alternating intrinsic (endogenous) electric fields as a means of controlling the speed and direction of microtubule-based transport. A specific model is proposed and analyzed in this paper. The model involves the rotational degrees of freedom of the C-termini of tubulin, their interactions and the coupling between elastic and dielectric degrees of freedom. Viscosity of the solution is also included and the resultant equation of motion is found as a nonlinear elliptic equation with dissipation. A particular analytical solution of this equation is obtained in the form of a kink whose properties are analyzed. It is concluded that this solution can be modulated by the presence of electric fields and hence may correspond to the observed behavior of motor protein transport along microtubules.     

Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.     

The Mechanism of Bactericidal Activity in Phagosomes of Neutrophils

Myeloperoxidase plays the key role in antimicrobial of phagocytes. This enzyme uses hydrogen peroxide and chloride to catalyze hypochlorous acid formation. HOCl is the most probable agent in the oxygen-dependent bactericidal activity in the phagocyte phagosome. Chlorination markers indicate HOCl generation in the quantities lethal for bacteria. Enzymatic assay for myeloperoxidase indicates proceeding of other reactions involved in bactericidal activity. Superoxide integrates many activities of this kind and is important for physiological function of myeloperoxidase. Elucidation of phagosomes biochemistry can help us to understand why certain pathogens survive in such unfavorable environment.     

Age-Dependent Changes in the Ultrastructure and in the Molecular Composition of Rat Brain Microtubules

An age-dependent increase of a cathepsin D-like protease activity that preferentially degrades high molecular weight microtubule-associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age-dependent changes in the relative amounts of MAPs and MT-associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of tau molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP-dependent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age-dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2-bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm-like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.     

Magnetic Separation of Phagosomes of Defined Age from Tetrahymena thermophila

ABSTRACT. We describe a new mass isolation procedure for both pure and stage-specific phagosomes from Tetrahymena thermophila . We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non-ingested particles, enabling us to follow the age-dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non-magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.     

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.     

Microtubules: a ring for the depolymerization motor

Newly discovered rings around microtubules, assembled from the Dam1 protein complex, may provide the dynamic linkage at microtubule ends for force generation coupled to microtubule depolymerization and polymerization.     

Evidence for Opposing Effects of Calmodulin on Cortical Microtubules

Microtubule integrity within the cortical array was visualized in detergent-lysed carrot (Daucus carota L.) protoplasts that were exposed to various exogenous levels of Ca2+ and calmodulin (CaM). CaM appears to help stabilize cortical microtubules against the destabilizing action of Ca2+/CaM complexes at low Ca2+ concentrations, but not at higher Ca2+ concentrations. The hypothesis that CaM interacts with microtubules at two different sites, determined by the concentration of Ca2+, is supported by the effects of the CaM antagonists N-(6-aminohexyl)-1-naphthalene-sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfanamide (20 [mu]M) and by affinity chromatography. Two classes of proteins were identified that interact with tubulin and bind to CaM. One class required Ca2+ for CaM binding, whereas the second class bound only when Ca2+ concentrations were low (<320 nM). Thus, CaM's ability to have two opposing effects upon microtubules may be regulated by the concentration of intracellular Ca2+ and its differential interactions with microtubule-associated proteins. Experimental manipulation of intracellular Ca2+ concentrations, as monitored by Indo-1, revealed that the effect of Ca2+ is specific to the cortical microtubules and does not affect actin microfilaments in these cells.     

Microtubules are necessary for lamellae retraction of Vero cells

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.     

Molecular Requirements for Sorting of the Chemokine Interleukin-8/CXCL8 to Endothelial Weibel-Palade Bodies

Sorting of proteins to Weibel-Palade bodies (WPB) of endothelial cells allows rapid regulated secretion of leukocyte-recruiting P-selectin and chemokines as well as procoagulant von Willebrand factor (VWF). Here we show by domain swap studies that the exposed aspartic acid in loop 2 (Ser⁴⁴-Asp⁴⁵-Gly⁴⁶) of the CXC chemokine interleukin (IL)-8 is crucial for targeting to WPB. Loop 2 also governs sorting of chemokines to α-granules of platelets, but the fingerprint of the loop 2 of these chemokines differs from that of IL-8. On the other hand, loop 2 of IL-8 closely resembles a surface-exposed sequence of the VWF propeptide, the region of VWF that directs sorting of the protein to WPB. We conclude that loop 2 of IL-8 constitutes a critical signal for sorting to WPB and propose a general role for this loop in the sorting of chemokines to compartments of regulated secretion.The regulated secretion of proteins from vascular endothelial cells provides a mechanism for their rapid delivery in response to secretagogues (1–4). The best characterized organelle for such secretion is the cigar-shaped Weibel-Palade body (WPB)³ that contains a number of proteins important in hemostasis and inflammation. For example, release of WPB is most likely involved in the early events of acute inflammation, since P-selectin stored in this compartment (5) mediates the tethering and rolling of leukocytes (6) and even appears to be the dominant selectin involved in ischemia/reperfusion injury (7, 8). Moreover, we and others have previously shown that the chemokines interleukin-8 (IL-8)/CXCL8 (9, 10) and eotaxin-3/CCL26 (11) can also be stored in WPB and hence are prime candidates for converting the selectin-mediated rolling of leukocytes into integrin-mediated firm adhesion. In this respect, WPB can be considered a “Swiss army knife of leukocyte recruitment,” able to rapidly deliver selectins and chemokines to the surface of endothelial cells that constitutively express the integrin ligands intercellular adhesion molecule-1 and -2.Targeting of proteins to compartments of regulated secretion is postulated to be an active process where proteins are segregated from the default route of exocytosis, the constitutive secretory pathway (12). The formation of WPB appears to be triggered by the expression of its main constituent, the 350-kDa hemostatic glycoprotein von Willebrand factor (VWF) (1, 13, 14). VWF entails a large propeptide (741 amino acids) that has been shown to be crucial for both the sorting of VWF (14, 15) and its multimerization (13, 14). Interaction of the propeptide and the mature part of VWF is moreover required for the formation of WPB in endothelial cells (16). Multimerization and organization of VWF and its propeptide into tubules takes place in the trans-Golgi network (TGN) and is followed by the formation of an extensive coat of AP-1/clathrin around the emerging organelles (17). Furthermore, it appears that other molecules can be targeted to WPB by interaction with VWF. For osteoprotegerin (18) and P-selectin (5, 19), this probably happens at the TGN level. IL-8 also binds to VWF under conditions mimicking those of the TGN, and subcellular fractionation analysis revealed that IL-8 is stored in a stoichiometric ratio to VWF, suggesting a direct molecular interaction (20).Mechanisms of chemokine sorting to the regulated secretory pathway are currently not well understood, but recent data from blood platelets point to a role for an exposed loop between the second and third β-strands of the molecule, referred to as loop 2 or the 40s loop, in the sorting of PF4/CXCL4, RANTES/CCL5, and NAP-2/CXCL7 to platelet α-granules (21). Specifically, the sorting of PF4 appears to depend on loop 2, featuring the residues ⁴⁵Leu-Lys-Asn-Gly⁴⁸ (21), and loops with similar three-dimensional structures are also found in RANTES and NAP-2, suggesting that this motif may be of general significance for chemokine sorting in platelets (21). In this regard, it may be of relevance that GROα (growth-related oncogene α)/CXCL1 contains a loop 2 sequence identical to that of PF4 and is found together with MCP-1 (monocyte chemotactic protein-1)/CCL2 in a recently characterized endothelial compartment for regulated secretion that we have designated type II granules of regulated secretion (11, 22).The aim of this study was to elucidate the molecular properties of IL-8 that enable its targeting to endothelial WPB. Chemokines have a highly similar tertiary structure, consisting of a typical Greek key structural motif with three consecutive β-strands forming a β-sheet stabilized by one or two disulfide bridges and a C-terminal α-helix inclined at a 45° angle to the β-sheet (Fig. 2, A and B) (23). This similarity in protein fold and the existence of endothelial cell-derived chemokines that are not sorted to WPB (11) make them ideal candidates for domain swap studies. By means of alanine mutations or chimerization with IP-10 (interferon-γ-induced protein-10), a chemokine not targeted to WPB (11), we demonstrated that loop 2 of IL-8, consisting of the residues Ser⁴⁴-Asp⁴⁵-Gly⁴⁶, is essential for sorting of the chemokine to WPB. Moreover, a chimera containing loop 2 and the α-helix from IL-8 on an IP-10 backbone sorted to WPB with the same efficiency as IL-8. The loop 2 area of IL-8 maintains a neutral net charge, and we propose that WPB targeting of IL-8 can occur when the overall charge is neutral or negative, whereas a positive charge is not tolerated. The fingerprint of the loop 2 region differs among chemokines and may confer specificity for sorting.Open in a separate windowFIGURE 2.Structural properties and distribution of IL-8 and IP-10 constructs and IL-8/IP-10 chimeras in HUVECs. A and B, IL-8 shares the characteristic secondary structure of most chemokines, with an N-terminal flexible region culminating in an N-loop region and a short 3₁₀-helix, followed by three anti-parallel β-strands and a C-terminal α-helix. Loop 2 is located between strands β-2 and β-3. IL-8 may form homodimers, in which case two β1-strands align to form an extended six-stranded β-sheet. Loop 2 of each monomer is exposed on opposite sides of the dimer. All central hydrophobic amino acid side chains making up the protein core connecting the β-sheet and α-helices of the two peptide chains are shown in ball-and-stick representations. The viewpoint in B is rotated 90° relative to A. C, transfected HUVECs were immunostained for a construct-derived HA tag (monoclonal antibody HA-7 or HA.11; green) and for VWF (red) as a marker of WPB. WT IL-8 colocalized with VWF, as demonstrated by the presence of yellow WPB (top). Mutation of Asp⁴⁵ in loop 2 of IL-8 to K (D45K) arrested WPB localization. As previously shown, IP-10 does not colocalize with VWF. However, residues 23–51 of IL-8 (Ch4), loop 2 of IL-8 in combination with loop 3 of IL-8 (Ch8), or the residues of loop 2 of IL-8 plus the C-terminal α-helix of IL-8 (Ch9) grafted to IP-10 restored sorting to WPB. The corner insets show high magnification of framed areas. Scale bar, 10 μm.     

Molecular and Functional Analyses of a Maize Autoactive NB-LRR Protein Identify Precise Structural Requirements for Activity

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.     

Cytochemical Nature and Fine Structure of the Gregarine Zeylanocystis burti Dissanaike, with Special Reference to Microfilaments, Microtubules and Movement

SYNOPSIS. The mature trophozoite of the acephaline gregarine Zeylanocystis burti Dissanaike, parasitic in the seminal vesicles of the Sri Lankan earthworm Pheretima peguana Rosa, was studied by cytochemical methods and by electron microscopy. Some observations were also made on gametocysts and oocysts. The trophozoite has a peculiar saucer shape, unlike other monocystid gregarines, and has marginal papillae with cytoplasmic hairs. Its fine structure conforms broadly to that of other gregarines, but differs with respect to its fibrillar organization and in some details of cytoplasmic organelle structure. The pellicle is composed of 2 parallel unit membranes elevated into a number of stumpy epicytary folds, more or less evenly distributed on both surfaces of the gregarine. Some of these are associated with adjacent accessory cells and may have a nutritive role. Bundles of fine microfilaments (5–6 nm) were detected both in the ectoplasm and deep in the endoplasm; these are possibly the main contractile elements (“myonemes”) involved in movement. Microtubules are larger (22–24 nm) and are found predominantly in papillae and cytoplasmic hairs, but extend also in small bundles beneath the pellicle—they appear to be more skeletal in nature. The significance of these findings is discussed in the light of recent work on the roles of microfilaments and microtubules in nonmuscle cells. Mitochondria are located superficially and have a complex organization. The endoplasmic reticulum is poorly developed, but ribosomes are abundant. Golgi lamellae-like membranes and vesicles akin to lysosomes were observed. Typical paraglycogen granules were found together with blobs of lipid and glycogen. The nucleus had a wrinkled envelope, a homogeneous matrix, and a spherical nucleolus. A variety of staining reactions and cytochemical tests were carried out. The distribution of lipids, polysaccharides, nucleic acids, calcium, and melanin were studied. Succinate dehydrogenase was detected in mitochondria, thiamine pyrophosphatase in Golgi bodies, and acid phosphatase in lysosomes. Golgi structures were found to be chemically very complex. Gametocysts and oocysts were associated with extraneous cells which probably contribute to the formation of their walls. The gametocyst wall is thin and consists of 2 membranes of the unit type. The oocyst wall is thicker and composed of 2 chemically different layers. Telolysosomes were seen in the disorganized residual cytoplasm within gametocysts.