Tracks for traffic: microtubules in the plant pathogen Ustilago maydis

Pathogenic development of the corn smut fungus Ustilago maydis depends on the ability of the hypha to grow invasively. Extended hyphal growth and mitosis require microtubules, as revealed by recent studies on the microtubule cytoskeleton. Surprisingly, hyphal tip growth involves only two out of 10 kinesins. Kinesin-3 is responsible for tip-directed (anterograde) endosome motility of early endosomes, which are thought to support hyphal elongation by apical membrane recycling. In addition, kinesin-3, together with kinesin-1 and myosin-5, appear to deliver secretory vesicles to the hyphal tip. Kinesin-1 also affects endosome motility by targeting cytoplasmic dynein to microtubule plus ends. This plus-end localization of dynein is essential for cell body-directed (retrograde) endosome motility, but also allows force generation during spindle elongation in mitosis. Furthermore, kinesin-1 and dynein participate in the organization of the microtubule array, thereby building their own network of tracks for intracellular motility. The recent progress in understanding microtubule-based processes in U. maydis has revealed an unexpected complexity of motor functions essential for the virulence of this pathogen. Further studies on structural and regulatory requirements for motor activity should help identify novel targets for fungicide development.     

Axonal transport of microtubules: the long and short of it

Recent studies on cultured neurons have demonstrated that microtubules are transported down the axon in the form of short polymers. The transport of these microtubules is bidirectional, intermittent, asynchronous, and occurs at the fast rate of known motors. The majority of the microtubule mass in the axon exists in the form of longer immobile microtubules. We have proposed a model called 'cut and run', in which the longer microtubules are mobilized by enzymes that sever them into shorter mobile polymers. In this view, the molecular motors that transport microtubules are not selective for short microtubules but rather impinge upon microtubules irrespective of their length. In the case of the longer microtubules, these motor-driven forces do not transport the microtubules in a rapid and concerted fashion but presumably affect them nonetheless. Here, we discuss the mechanisms by which the short microtubules are transported and suggest possibilities for how analogous mechanisms may align and organize the longer microtubules and functionally integrate them with each other and with the actin cytoskeleton.     

Effects of dynactin disruption and dynein depletion on axonal microtubules

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.     

Viral strategies for triggering and manipulating mitophagy

Viral infection causes many physiological alterations in the host cell, and many of these alterations can affect the host mitochondrial network, including mitophagy induction. A substantial amount of literature has been generated that advances our understanding of the relationship between mitophagy and several viruses. Some viruses trigger mitophagy directly, and indirectly and control the mitophagic process via different strategies. This enables viruses to promote persistent infection and attenuate the innate immune responses. In this review, we discuss the events of virus-regulated mitophagy and the functional relevance of mitophagy in the pathogenesis of viral infection and disease.

Abbreviation: ATG: autophagy related; BCL2L13: BCL2 like 13; BNIP3L/NIX: BCL2 interacting protein 3 like; CL: cardiolipin; CSFV: classical swine fever virus; CVB: coxsackievirus B; DENV: dengue virus; DNM1L: dynamin 1 like; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; HPIV3: human parainfluenza virus 3; HSV-1: herpes simplex virus type 1; IMM: inner mitochondrial membrane; IAV: influenza A virus; IFN: interferon; IKBKE/IKKε: inhibitor of nuclear factor kappa B kinase subunit epsilon; LUBAC: linear ubiquitin assembly complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MeV: measles virus; MAVS: mitochondrial antiviral signaling protein; MFF: mitochondria fission factor; NLRP3: NLR family pyrin domain containing 3; NDV: Newcastle disease virus; NR4A1: nuclear receptor subfamily 4 group A member 1; OMM: outer mitochondrial membrane; OPA1: OPA1, mitochondrial dynamin like GTPase; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; PHB2: prohibitin 2; PRRSV: porcine reproductive and respiratory syndrome virus; PRRs: pattern-recognition receptors; RLRs: RIG-I-like receptors; ROS: reactive oxygen species; RIPK2: receptor interacting serine/threonine kinase 2; SESN2: sestrin 2; SNAP29: synaptosome associated protein 29; STX17: syntaxin 17; TGEV: transmissible gastroenteritis virus; TUFM: Tu translation elongation factor, mitochondrial; TRAF2: TNF receptor associated factor 2; TRIM6: tripartite motif containing 6; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; VZV: varicella-zoster virus     

Microtubule-associated proteins and motors required for ectopic microtubule array formation in Saccharomyces cerevisiae

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.     

Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons

Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC) ‬‬‬‬‬‬‬

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.     

Cytoskeletal elements and intracellular transport

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.     

Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in oocytes

In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball‐like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore–microtubule attachments in fine‐tuning acentrosomal spindle bipolarity.     

Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag

Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.

Structured summary

MINT-7014185: Gag (uniprotkb:P05888) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7014239: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RelA (uniprotkb:Q04206), RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014046: gag (uniprotkb:P05888), SOCS1 (uniprotkb:O15524) and tubulin alpha (uniprotkb:Q13748) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014269: tubulin alpha (uniprotkb:Q13748) physically interacts (MI:0218) with Gag (uniprotkb:P05888) by anti tag coimmunoprecipitation (MI:0007)MINT-7014036: tubulin alpha (uniprotkb:Q13748) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014201: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014257: Gag (uniprotkb:P05888) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7014221: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with Gag (uniprotkb:P05888), elongin C (uniprotkb:Q15370), elongin B (uniprotkb:Q15369), SOCS1 (uniprotkb:O15524) and RBX1 (uniprotkb:P62877) by pull-down (MI:0096)     

Dynamitin mutagenesis reveals protein-protein interactions important for dynactin structure

Dynactin is a highly conserved, multiprotein complex that works in conjunction with microtubule-based motors to power a variety of intracellular motile events. Dynamitin (p50) is a core element of dynactin structure. In the present study, we use targeted mutagenesis to evaluate how dynamitin's different structural domains contribute to its ability to self-associate, interact with dynactin and assemble into a complex with its close binding partner, p24. We show that these interactions involve three distinct structural elements: (i) a previously unidentified dimerization motif in the N-terminal 100 amino acids, (ii) an α-helical motif spanning aa 106–162 and (iii) the C-terminal half of the molecule (aa 213–406), which is predicted to fold into an antiparallel α-helix bundle. The N-terminal half of dynamitin by itself is sufficient to disrupt dynactin, although very high concentrations are required. The ability of mutations in dynamitin's interaction domains to disrupt dynactin in vitro was found to correlate with their inhibitory effects when expressed in cells. We determined that the dynactin subunit, p24, governs dynamitin oligomerization by binding dynamitin along its length. This suppresses aberrant multimerization and drives formation of a protein complex that is identical to the native dynactin shoulder.