Control Of Microtubule Dynamics Research Articles

Kinetic Characterization of Rice Plant Specific Kinesin E11 using Fluorescent ATP Analogue

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures.In this study, we characterized rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E. coli expression system and purified with Co-chelate column in order to characterize biochemical and ATPase kinetic properties. The fluorescent ATP analogues, Mant-ATP was employed for the kinetic characterization. We have successfully observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters of initial binding of Mant-ATP to E11 and release of Mant-ADP from E11 were analyzed by monitoring the FRET using stopped flow apparatus and compared with other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are slower than those of other rice plant specific kinesin.

Katanin Activity is Regulated by Common Cellular Components

Proper organization of the microtubule cytoskeletal network is required to perform necessary cellular functions. Network organization is achieved through remodeling by microtubule associated proteins (MAPs) that control microtubule dynamics by selectively stabilizing and destabilizing microtubules. Stabilizing MAPs are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule severing enzyme, is a AAA+ enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using single molecule biophysics techniques in vitro we investigate how katanin and its severing activity can be regulated by the type of microtubule, free tubulin, ATP concentration, and the neuronal MAP tau isoform 4RL. This work provides evidence that katanin activity is regulated by common cellular contents that will ultimately affect microtubule organization in cells.

Mitotic regulation of the mitotic centromere-associated kinesin

Deregulated mitotic centromere-associated kinesin (MCAK) is associated with cancer invasiveness, metastasis and poor prognosis of patients. It is thus important to delineate the regulatory mechanisms responsible for regulating MCAK's turnover. MCAK depolymerizes microtubules, controls microtubule dynamics and has been tightly linked to the correction of fail-attachments between kinetochores and microtubules in mitosis. Its activity and localization are regulated via phosphorylation by important mitotic kinases. The exact picture of MCAK regulation by these kinases during mitosis remains elusive and needs to be clarified. We show here that S621 in the C-terminal domain is the major site for phosphorylation of MCAK by Polo-like kinase 1 (Plk1). Phosphorylation at this location does not directly affect its microtubule depolymerization activity but rather likely promotes recognition of MCAK by the Cdc20 subunit of APC/C leading to its proteasome dependent degradation at the transition from metaphase to anaphase. However, loss of Plk1 phosphorylation on S621 indirectly enhances its depolymerization activity in vivo by preventing MCAK's targeting for degradation, resulting in an accumulation of MCAK protein. By contrast, phosphomimetic MCAK S621D is constitutively targeted for degradation leading to reduced levels of protein and a consequent increase in amount of polymerized tubulin and decreased inter-centromere distance in cells expressing this form of MCAK. Finally, interfering with this phosphorylation results in defects in spindle formation as well as in chromosome alignment, likely due to the lack of regulation of MCAK protein levels during mitosis.

Promoting microtubule assembly: A hypothesis for the functional significance of the +TIP network

Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.

DOCK5 functions as a key signaling adaptor that links FcεRI signals to microtubule dynamics during mast cell degranulation.

Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcεRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcεRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3β. When DOCK5-Nck2-Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.

Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition.

Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice is associated with developmental retardation and embryonic lethality. Here we present a family with a novel neuromuscular condition. Genetic analyses show a heterozygous duplication resulting in reduced MACF1 gene product. The functional consequence is affected motility observed as periodic hypotonia, lax muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances. These findings suggest that changes in the MACF1 gene is implicated in this neuromuscular condition, which is an important observation since MACF1 has not previously been associated with any human disease and thus presents a key to understanding the essential nature of this gene.

TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes.

Chromosomal instability can arise from defects in chromosome–microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1's role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome–microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore–microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.

Stable Microtubule Subsets Facilitate Early HIV-1 Infection

The density of the host cytoplasm presents pathogens such as HIV-1 with a major impediment to reaching the nucleus. As such, they must exploit host cytoskeletal networks to facilitate their movement.1 Within host cells, microtubule (MT) filaments act as “highways” for the intracellular transport of cargoes mediated by MT-associated motor proteins.2 While it has been established that the MT motor dynein mediates inward movement of HIV-1 to the nucleus,3 studies using MT depolymerizing agents such as nocodazole have produced seemingly conflicting suggestions that MTs are not important for HIV-1 infection. Recent findings relating to the role of specialized stable MT subsets now offer a potential explanation. MTs form polarized filaments; their minus ends are anchored in a perinuclear MT organizing center (MTOC) while their plus ends grow outward to explore or “sense” the intracellular environment and form connections to the cell periphery. As such, in many cell types MTs are highly dynamic, undergoing rapid phases of growth and collapse through the addition and loss of tubulin subunits primarily at the plus end. MT stability is controlled by highly specialized plus-end-binding proteins, called+TIPs. Central to this is the end-binding protein EB1, which recognizes the MT plus end and recruits other+TIPs that control MT dynamics. In response to specific signals, subsets of MTs become stabilized and form long-lived filaments that acquire distinguishing posttranslational tubulin modifications such as detyrosination and acetylation. Although these modifications do not seem to impart stability per se, they are thought to enable selective recognition of stable MT subsets by specific MT motor proteins. As such, these filaments can act as specialized tracks for vesicle trafficking to facilitate processes such as cell polarization. A notable feature of these long-lived MT subsets is their relative insensitivity to MT depolymerizing agents, but their potential contribution to HIV-1 infection has until now gone largely unstudied. We recently found that HIV-1 infection rapidly induces MT stabilization in a range of human cell types,4 including natural target cells such as T cells, microglia, and primary macrophages (Fig. 1). This stabilization appears to occur very soon after viral entry and involves viral targeting of EB1 and the EB1-binding+TIP, Kif4. MT stabilization continues to increase as infection progresses, potentially as more virus enters the cell, triggered by viral proteins released during uncoating or by the activation of signal pathways that regulate MT stability, something we are currently studying. Viral particles also exhibited significant localization to stable MTs and when stable MT formation was blocked using either RNA interference (RNAi) or dominant negative approaches infection was potently suppressed, with viral particles failing to reach the nucleus efficiently. These findings suggest that HIV-1 induces the formation of specialized stable MT subsets early after entry in order to facilitate translocation of viral particles to the nucleus. The importance of these stable MT subsets to early HIV-1 infection offers an explanation for why dynein is important for infection yet MT depolymerizing agents have little effect, and identifies new factors to explore as potential therapeutic targets to treat HIV-1. FIG. 1. HIV-1 induces stable microtubule (MT) formation in primary human macrophages. Primary human macrophages were mock infected (inset image lower left) or infected with pseudotyped HIV-1 and then fixed and stained for tyrosinated (red) and acetylated (green) ...

Hoxb1b controls oriented cell division, cell shape and microtubule dynamics in neural tube morphogenesis

Hox genes are classically ascribed to function in patterning the anterior-posterior axis of bilaterian animals; however, their role in directing molecular mechanisms underlying morphogenesis at the cellular level remains largely unstudied. We unveil a non-classical role for the zebrafish hoxb1b gene, which shares ancestral functions with mammalian Hoxa1, in controlling progenitor cell shape and oriented cell division during zebrafish anterior hindbrain neural tube morphogenesis. This is likely distinct from its role in cell fate acquisition and segment boundary formation. We show that, without affecting major components of apico-basal or planar cell polarity, Hoxb1b regulates mitotic spindle rotation during the oriented neural keel symmetric mitoses that are required for normal neural tube lumen formation in the zebrafish. This function correlates with a non-cell-autonomous requirement for Hoxb1b in regulating microtubule plus-end dynamics in progenitor cells in interphase. We propose that Hox genes can influence global tissue morphogenesis by control of microtubule dynamics in individual cells in vivo.

CAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

CFEOM1-Associated Kinesin KIF21A Is a Cortical Microtubule Growth Inhibitor

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/β1 and the components of the LL5β-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

MTB-3, a Microtubule Plus-End Tracking Protein (+TIP) of Neurospora crassa

The microtubule (MT) “plus end” constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called “MT plus-end tracking proteins” (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

The Kinesin-2 Family Member KIF3C Regulates Microtubule Dynamics and Is Required for Axon Growth and Regeneration

Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells

MAP1B, a structural microtubule (MT)-associated protein highly expressed in developing neurons, plays a key role in neurite and axon extension. However, not all molecular mechanisms by which MAP1B controls MT dynamics during these processes have been revealed. Here, we show that MAP1B interacts directly with EB1 and EB3 (EBs), two core 'microtubule plus-end tracking proteins' (+TIPs), and sequesters them in the cytosol of developing neuronal cells. MAP1B overexpression reduces EBs binding to plus-ends, whereas MAP1B downregulation increases binding of EBs to MTs. These alterations in EBs behaviour lead to changes in MT dynamics, in particular overstabilization and looping, in growth cones of MAP1B-deficient neurons. This contributes to growth cone remodelling and a delay in axon outgrowth. Together, our findings define a new and crucial role of MAP1B as a direct regulator of EBs function and MT dynamics during neurite and axon extension. Our data provide a new layer of MT regulation: a classical MAP, which binds to the MT lattice and not to the end, controls effective concentration of core +TIPs thereby regulating MTs at their plus-ends.

Tubulin Polymerization Promoting Protein 1 (Tppp1) Phosphorylation by Rho-associated Coiled-coil Kinase (Rock) and Cyclin-dependent Kinase 1 (Cdk1) Inhibits Microtubule Dynamics to Increase Cell Proliferation

Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

Compartimentation et plasticité du réseau microtubulaire

Cytoplasm organization greatly depends on the cytoskeleton and especially on microtubules. Their multiple roles comprise for instance long-distance vesicular traffic or the organization of several signalling pathways. A variety of cellular functions require highly dynamic microtubules, which alternate between growing and shrinking phases. Meanwhile, other functions use stable microtubules, in which tubulin often bears multiple post-translational modifications like acetylation. Recent progress has been made in understanding some molecular mechanisms that control microtubule dynamics or tubulin acetylation. These mechanisms reveal the high plasticity of microtubules and point out the importance of their compartmentalization at structural and functional levels.

Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration

The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels

Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.