Novel synthesis of ClF(6)(+) and BrF(6)(+) salts.

For a compound in a given oxidation state, its oxidizing strength increases from its anion to the neutral parent molecule to its cation. Similarly, an anion is more easily oxidized than its neutral parent molecule, which in turn is more easily oxidized than its cation. This concept was systematically exploited in our search for new superoxidizers. Transition metal fluoride anions were prepared in their highest known oxidation states by high temperature/high pressure fluorinations with elemental fluorine and subsequently converted to their more strongly oxidizing cations by a displacement reaction with a strong Lewis acid. The application of this principle resulted in new syntheses for ClF(6)(+)AsF(6)(-) and BrF(6)(+)AsF(6)(-) using the highly reactive and thermally unstable NiF(3)(+) cation that was prepared from the reaction of the NiF(6)(2)(-) anion with AsF(5) in anhydrous HF. Attempts to prepare the known KrF(+) and ClO(2)F(2)(+) cations and the yet unknown XeF(7)(+) cation by the same method were unsuccessful. The results from this and previous studies show that NiF(3)(+) is a stronger oxidative fluorinator than PtF(6), but whether its oxidizing strength exceeds that of KrF(+) remains unclear. Its failure to oxidize Kr to KrF(+) might have been due to unfavorable reaction conditions. Its failure to oxidize ClO(2)F to ClO(2)F(2)(+), in spite of its favorable oxidizer strength, is attributed to the high Lewis basicity of ClO(2)F which results in a rapid displacement reaction of NiF(3)(+) by ClO(2)F, thus generating the weaker oxidizer NiF(4) and the more difficult to oxidize substrate ClO(2)(+). Therefore, the general applicability of this approach appears to be limited to substrates that exhibit a weaker Lewis basicity than the neutral transition metal parent molecule. Compared to KrF(+)- or PtF(6)-based oxidations, the NiF(3)(+) system offers the advantages of commercially available starting materials and higher yields, but product purification can be more difficult and tedious than for KrF(+).

Microtubules don and doff their caps: dynamic attachments at plus and minus ends.

Microtubule plus end dynamics, as well as interactions with the cell cortex and internal organelles, may be mediated by a 'plus end complex' of interacting proteins. Recent results suggest that centrosomes with different microtubule-releasing and anchoring properties underlie the development of various microtubule arrays.

Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles.

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150(Glued)) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.

Real time imaging reveals a peroxisomal reticulum in living cells.

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

Dynactin increases the processivity of the cytoplasmic dynein motor.

Cytoplasmic dynein supports long-range intracellular movements of cargo in vivo but does not appear to be a processive motor protein by itself. We show here that the dynein activator, dynactin, binds microtubules and increases the average length of cytoplasmic-dynein-driven movements without affecting the velocity or microtubule-stimulated ATPase kinetics of cytoplasmic dynein. Enhancement of microtubule binding and motility by dynactin are both inhibited by an antibody to dynactin's microtubule-binding domain. These results indicate that dynactin acts as a processivity factor for cytoplasmic-dynein-based motility and provide the first evidence that cytoskeletal motor processivity can be affected by extrinsic factors.

Motors, clutches and brakes for membrane traffic: a commemorative review in honor of Thomas Kreis.

Membranous organelles interact with a wide variety of cytoskeletal proteins that allow them to be organized into dynamic, yet stable, structures with distinct subcellular addresses. This review provides an up-to-date summary of the motor enzymes and membrane-microtubule crosslinking proteins that have been implicated in this process, and discusses the potential impact membrane anchoring may have on cellular architecture.

Actin-related proteins.

Actin-related proteins (Arps) participate in a diverse array of cellular processes. They modulate assembly of conventional actin, contribute to microtubule-based motility catalyzed by dynein, and serve as integral components of large protein complexes required for gene expression. We highlight here recent work aimed at understanding the roles played by Arps in each of these processes.

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the arp1 minifilament pointed end.

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

Dynactin is required for microtubule anchoring at centrosomes.

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein-dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein-dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, gamma tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin-cargo interactions.

C(6)F(5)XeCl and

Xenon(II) chlorine compounds can be obtained as the isolable organo derivatives C(6)F(5)XeCl and [(C(6)F(5)Xe)(2)Cl][AsF(6)] (whose cation is depicted) in 85 and 91 % yield, respectively. These compounds decompose vigorously at 36 degrees C and 100 degrees C, respectively, leading to the formation of C(6)F(5)Cl and Xe gas and of C(6)F(5)Cl, C(6)F(6), and [C(6)F(5)Xe][AsF(6)], respectively.

Membrane motors.

Research over the past 18 months has revealed that many membranous organelles move along both actin filaments and microtubules. It is highly likely that the activity of the microtubule motors, myosins and static linker proteins present on any organelle are co-ordinately regulated and that this control is linked to the processes of membrane traffic itself.

Self-regulated polymerization of the actin-related protein Arp1.

The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments.

Role for microtubules in centrosome doubling in Chinese hamster ovary cells.

The centrosome must be replicated once, and only once, during each cell cycle. To achieve this somatic cells need to synthesize centrosome proteins, target those centrosome proteins to the parental centrosome, and then assemble the centrosome subunits into a functional organelle. The mechanisms that underlie each of these processes are not known. Studies were performed to investigate whether cellular microtubules are involved in centrosome doubling events. For these experiments, CHO cells were arrested in either hydroxyurea (HU) alone or in HU plus a microtubule inhibitor for 3640 h. The cells then were induced to enter mitosis and the numbers of spindle poles/centrosomes were counted following processing of the cells for immunofluorescence microscopy using anticentrosome antiserum. These studies demonstrated that centrosome replication events occurred in cells arrested with either HU alone or HU and taxol while centrosome replication did not occur in cells treated with HU and either nocodazole or colcemid. Immunoblot analysis determined that centrosome proteins were synthesized in HU/nocodazole-arrested cells and demonstrated that the role of microtubules in the centrosome replication process is not to ensure the synthesis of centrosome subunits. Rather, our results suggest that microtubules may be involved in the transport/targeting of centrosome subunits to the parental centrosome during duplication events. For microtubules to contribute to the transport of centrosome subunits during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were performed and it was determined that the centrosome proteins, though overproduced under these conditions, remained soluble in HU/nocodazole-treated cells and co-pelleted with taxol-stabilized microtubules in the presence of GTP and AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PCM-1, with microtubules could be inhibited by pre-incubation of extracts with antibodies against dynactin. Together, these data suggest that during centrosome replication in somatic mammalian cells, PCM-1, and perhaps other centrosome components, are targeted to the centrosome via transport along microtubules by motor complexes that include dynein/dynactin.

Expression of PEX11beta mediates peroxisome proliferation in the absence of extracellular stimuli.

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.

Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment.

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.

Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct.

We investigated whether the motor protein cytoplasmic dynein and dynactin, a protein complex thought to link dynein with vesicles, are present in rat renal collecting ducts and associated with aquaporin-2 (AQP2)-bearing vesicles. Immunoblotting demonstrated cytoplasmic dynein heavy and intermediate chains in kidney, with relative expression levels of inner medulla > outer medulla > cortex. In addition to being present in cytoplasmic fractions, dynein was abundant in membrane fractions enriched for intracellular vesicles. Dynactin was also abundant in membrane fractions enriched for intracellular vesicles. Furthermore, both dynactin and dynein were present in vesicles specifically immunoisolated using anti-AQP2 antibodies. Immunocytochemistry revealed labeling for dynein in the collecting duct principal cells with a pattern consistent with labeling of intracellular vesicles. Moreover, quantitative double immunogold labeling confirmed colocalization of AQP2 and dynein in the same vesicles at the electron microscopic level. Thus the microtubule-associated motor protein dynein and the associated dynactin complex are present in rat renal collecting duct principal cells and are associated with intracellular vesicles, including those bearing AQP2, consistent with the view that dynein and dynactin are involved in vasopressin-regulated trafficking of AQP2-bearing vesicles.

ER-to-Golgi transport visualized in living cells.

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.