Plus Ends Of Microtubules Research Articles

The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization

Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in β-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant β-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of β-tubulin and the resulting destabilization of cortical microtubules.

Abstract 349: Eribulin induces cortical localization of E-cadherin, p120-catenin and beta-catenin in breast cancer cells

Abstract Eribulin provides a survival advantage for breast cancer patients, particularly those with triple-negative and HER2-negative subtypes.1,2 Mechanistically, eribulin was shown to reverse epithelial-to-mesenchymal transition (EMT) in breast cancer models.3 E-cadherin is a central regulator responsible for maintenance of the epithelial phenotype and loss of cortical E-cadherin and its downstream signaling is an early and significant event in EMT. Short term treatment of HCC1937 breast cancer cells with eribulin initiated cortical E-cadherin localization in manner different from other microtubule targeting agents. Cortical E-cadherin plays essential roles in cell adhesion and in regulating the Wnt pathway, whose aberrant activity is implicated in EMT. At the adherens junctions p120-catenin and β-catenin bind to E-cadherin, an interaction facilitated by microtubules. Cortical sequestration of p120- and β-catenin at the adherens junctions stabilizes these complexes and inhibits their ability to stimulate signaling pathways that contribute to EMT. We tested whether eribulin, which binds to the plus ends of microtubules, promotes EMT reversal by influencing p120- and β-catenin localization and activity. Our results show that eribulin induces cortical localization of p120-catenin and β-catenin with E-cadherin in HCC1937 cells, consistent with the formation of normal adherens junctions. Eribulin also stimulated the phosphorylation of Ser33/37 and Thr41 on β-catenin which promotes proteasomal degradation, which would be expected to further inhibit oncogenic signaling. Collectively, our results suggest that eribulin contributes to reversal of EMT through modulation of downstream signaling initiated by cortical localization of p120- and β-catenin at the adherens junctions. Funding for this work was provided by Eisai Inc. 1. Cortes J., et al., Lancet 377:914-923, 2011 2. Twelves, C., et al., Breast Cancer Res Treat 148:553-561, 2014 3. Yoshida, T., et al., Br J of Cancer 110: 1497-1505, 2014 Citation Format: Alison D. Clark, Nicholas F. Dybdal-Hargreaves, Susan L. Mooberry. Eribulin induces cortical localization of E-cadherin, p120-catenin and beta-catenin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 349. doi:10.1158/1538-7445.AM2017-349

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation.

During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation.

EB-Family Proteins: Functions and Microtubule Interaction Mechanisms.

Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.

Abstract 4169: A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer

Abstract The microtubule (MT) cytoskeleton is one of the most validated therapeutic targets in clinical oncology, as evidenced by the wide clinical activity of taxanes and other MT inhibitors. However, patients with intrinsic drug resistance do not benefit from taxane chemotherapy, such as patients with diffuse gastric cancer (GC). Currently, the molecular mechanisms underlying clinical taxane resistance remain poorly elucidated. Using a panel of GC cell lines with intrinsic sensitivity or resistance to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular accumulation of the drug and in the absence of tubulin mutations or altered tubulin isotype expression. Herein, we show for the first time that the resistant cells expressed a novel, short isoform of the MT plus-end binding protein, CLIP170 (CLIP1). CLIP1 binds to the plus-ends of microtubules (+TIPs) and regulates dynein-mediated MT-based trafficking and the tubulin tyrosination cycle. We showed by both mass-spec proteomics and 5’-RACE that the short CLIP1 isoform (CLIP1S) lacks the first 150 amino acids, thus, missing the first Cap-Gly (Cytoskeleton-Associated Protein-Glycine) domain, which is required for proper +TIP localization. Indeed, confocal microscopy experiments showed that CLIP1S was mislocalized to the microtubule lattice in contrast to the canonical comet-like pattern of CLIP1 seen in taxane sensitive cell lines. CLIP1S expression was specifically correlated with taxane resistance (docetaxel, cabazitaxel) as no correlation was observed with other DNA-damaging agents. Since, CLIP1S expression has never been reported before, to establish causation, we stably knocked down CLIP1 and CLIP1S in taxane-sensitive and resistant cells, respectively. CLIP1SKD entirely reversed taxane-resistance (~300 fold) while CLIP1KD had no effect in the taxane-sensitive cell lines. These data suggest a gain-of-function of CLIP1S that leads to taxane resistance. To study the binding kinetics of taxanes to MTs in the presence or absence of CLIP1S we performed live cell imaging of native cytoskeletons using fluorescently-labeled paclitaxel (Flutax). Our data revealed significantly faster dissociation rates of Flutax from MTs in the resistant cells, indicating transient interaction with MTs. Taxane binding to MTs is a two-step process. First taxanes bind to the MT-pore low affinity surface site, which then facilitates access to the high affinity luminal site. Using a small molecule that binds only to MT-pore site, we showed that we showed that taxol binding to the pore is significantly slower in resistant cells expressing CLIP1S, which together with mislocalization of this variant along the MT lattice suggests that it obstructs access to the MT-pore thus restricting entry of taxane into the lumen of microtubules. This finding will have profound implications for taxane resistance as well as microtubule biology broadly. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Isabel Barasoain, Jose Fernando Diaz, Manish A. Shah, Paraskevi Giannakakou. A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2017-4169

The ARM Domain of ARMADILLO-REPEAT KINESIN 1 is Not Required for Microtubule Catastrophe But Can Negatively Regulate NIMA-RELATED KINASE 6 in Arabidopsis thaliana.

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.

Purification and characterisation of the fission yeast Ndc80 complex

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.

An Potogenentic Toolkit for Reversible Labeling and Remote Manipulation of Cytoskeleton In Situ

Most of the current methodologies to visualize the cytoskeleton are irreversible and can only provide a static picture of cytoskeleton. These methods often involve the fixation of cells or permanently perturb the behaviors of cytoskeleton. To overcome this obstacle, we designed a set of optogenetic tools to reversibly label and manipulate cytoskeleton dynamics in living cells by harnessing the power of light. These genetically-encoded small optical tools were demonstrated to i) instantly and reversibly photo-label actin, microtubule and the plus-end of microtubule without perturbing their normal functions, ii) manipulate the dynamics of microtubule following stimulation with light emitting at varying wavelengths; and iii) drive the target protein or organelle towards either ends of MTs to recruit effectors and control the downstream signaling pathways. These noninvasive optical tools provide new opportunities to remotely monitor and perturb the cytoskeleton, and can also be applied in drug screening to identify potential adverse effects of drug candidates on cytoskeleton. (Supported by the National Institutes of Health (GM112003) and the Welch Foundation (BE-1913 to Y.Z.), and a China Scholarship Council award to Q.Z.)

Dissection of Microtubule Mediated Mechanical Forces in Mitosis

During cell division, dynamic microtubule plus-ends associate with kinetochores to align and stretch duplicated sister chromatids at the center of a metaphase mitotic spindle, generating a tension force that is thought to be important for ensuring the fidelity of chromosome segregation during mitosis. However, the number of microtubules that are associated with the kinetochore varies widely between organisms. A mechanistic understanding for how tension may scale with increasing numbers of kinetochore microtubule attachments is unknown. In this work, we manipulated the average number of microtubules per kinetochore within a single organism, and found that average tension was coordinately increased with increasing numbers of kinetochore microtubules. Computational modeling of our experimental results suggested that tension-dependent microtubule dynamics combined with increasing numbers of microtubule attachments may provide a seamless method for scaling up tension magnitudes from a simple organism such as yeast to a more complex organism with multiple microtubules per kinetochore.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

The MgcRacGAP SxIP motif tethers Centralspindlin to microtubule plus ends in Xenopus laevis.

Centralspindlin, a complex of the kinesin-6-family member MKLP1 and MgcRacGAP (also known as Kif23 and Racgap1, respectively), is required for cytokinesis and cell-cell junctions. During anaphase, Centralspindlin accumulates at overlapping central spindle microtubules and directs contractile ring formation by recruiting the GEF Ect2 to the cell equator to activate RhoA. We found that MgcRacGAP localized to the plus ends of equatorial astral microtubules during cytokinesis in Xenopus laevis embryos. How MgcRacGAP is stabilized at microtubule plus ends is unknown. We identified an SxIP motif in X. laevis MgcRacGAP that is conserved with other proteins that bind to EB1 (also known as Mapre1), a microtubule plus-end tracking protein. Mutation of the SxIP motif in MgcRacGAP resulted in loss of MgcRacGAP tracking with EB3 (also known as Mapre3) on growing microtubule plus ends, abnormal astral microtubule organization, redistribution of MgcRacGAP from the contractile ring to the polar cell cortex, and mislocalization of RhoA and its downstream targets, which together contributed to severe cytokinesis defects. Furthermore, mutation of the MgcRacGAP SxIP motif perturbed adherens junctions. We propose that the MgcRacGAP SxIP motif is functionally important both for its role in regulating adherens junction structure during interphase and for regulating Rho GTPase activity during cytokinesis.

A TIRF microscopy assay to decode how tau regulates EB's tracking at microtubule ends.

Tau is a major microtubule-associated protein (MAP) mainly expressed in the brain. Tau binds the lattice of microtubules and favors their elongation and bundling. Recent studies have shown that tau is also a partner of end-binding proteins (EBs) in neurons. EBs belong to the protein family of the plus-end tracking proteins that preferentially associate with the growing plus-ends of microtubules and control microtubule end behavior and anchorage to intracellular organelles. Reconstituted cell-free systems using purified proteins are required to understand the precise mechanisms by which tau influences EB localization on microtubules and how the concerted activity of these two MAPs modulates microtubule dynamics. We developed an in vitro assay combining TIRF microscopy and site-directed mutagenesis to dissect the interaction of tau with EBs and to study how this interaction affects microtubule dynamics. Here, we describe the detailed procedures to purify proteins (tubulin, tau, and EBs), prepare the samples for TIRF microscopy, and analyze microtubule dynamics, and EB binding at microtubule ends in the presence of tau.

Phosphorylation of EB1 regulates the recruitment of CLIP-170 and p150glued to the plus ends of astral microtubules.

Phosphorylation of end-binding protein 1 (EB1), a key member of microtubule plus end-tracking proteins (+TIPs), by apoptosis signal-regulating kinase 1 (ASK1) has been demonstrated to promote the stability of astral microtubules during mitosis by stimulating the binding of EB1 to microtubule plus ends. However, the roles of other members of the +TIPs family in ASK1/EB1-mediated regulation of astral microtubules are unknown. Herein, we show that ASK1-mediated phosphorylation of EB1 enhances the localization of cytoplasmic linker protein 170 (CLIP-170) and p150glued to the plus ends of astral microtubules. Depletion of ASK1 or expression of phospho-deficient or phospho-mimetic EB1 mutants results in changes in the levels of plus-end localized CLIP-170 or p150glued. Mechanistic studies reveal that EB1 phosphorylation promotes its interactions with CLIP-170 and p150glued, thereby recruiting these +TIPs to microtubules. Structural analysis suggests that serine-40 is the primary phosphorylation site on EB1 that exerts these effects. Together, these findings provide novel insight into the molecular mechanisms that regulate the interactions of EB1 with other +TIPs.

Phosphorylation of SKAP by GSK3β ensures chromosome segregation by a temporal inhibition of Kif2b activity.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent study shows SKAP is an EB1-dependent, microtubule plus-end tracking protein essential for kinetochore oscillations during mitosis. Here we show that phosphorylation of SKAP by GSK3β regulates Kif2b depolymerase activity by competing Kif2b for microtubule plus-end binding. SKAP is a bona fide substrate of GSK3β in vitro and the phosphorylation is essential for an accurate kinetochore-microtubule attachment in cells. The GSK3β-elicited phosphorylation sites were mapped by mass spectrometry and the phosphomimetic mutant of SKAP can rescue the phenotype of chromosome missegregation in SKAP-suppressed cells. Importantly, GSK3β-elicited phosphorylation promotes SKAP binding to Kif2b to regulate its depolymerase activity at the microtubule plus-ends. Based on those findings, we reason that GSK3β-SKAP-Kif2b signaling axis constitutes a dynamic link between spindle microtubule plus-ends and mitotic chromosomes to achieve faithful cell division.

ASK1 controls spindle orientation and positioning by phosphorylating EB1 and stabilizing astral microtubules

Orientation and positioning of the mitotic spindle are involved in dictating cell division axis and cleavage site, and play important roles in cell fate determination and tissue morphogenesis. However, how spindle movement is controlled to achieve a defined alignment within the dividing cell is not fully understood. Here, we describe an unexpected role for apoptosis signal-regulating kinase 1 (ASK1) in regulating spindle behavior. We find that ASK1 is required for proper mitotic progression and daughter cell adhesion to the substratum. ASK1 interacts with end-binding protein 1 (EB1) and phosphorylates EB1 at serine 40, threonine 154 and threonine 206, enhancing its binding to the plus ends of astral microtubules. Consequently, astral microtubules are stabilized and therefore capable of mediating spindle interaction with the cell cortex, a requirement for spindle movement. These findings reveal a previously undiscovered function of ASK1 in cell division by regulating spindle orientation and positioning, and point to the importance of protein phosphorylation in the regulation of spindle behavior.

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

Xenopus TACC2 is a microtubule plus end-tracking protein that can promote microtubule polymerization during embryonic development.

Microtubule dynamics is regulated by plus end-tracking proteins (+TIPs), which localize to the plus ends of microtubules (MTs). We previously showed that TACC1 and TACC3, members of the transforming acidic coiled-coil protein family, can act as +TIPs to regulate MT dynamics in Xenopus laevis Here we characterize TACC2 as a +TIP that localizes to MT plus ends in front of EB1 and overlapping with TACC1 and TACC3 in multiple embryonic cell types. We also show that TACC2 can promote MT polymerization in mesenchymal cells but not neuronal growth cones, thus displaying cell-type specificity. Structure-function analysis demonstrates that the C-terminal region of TACC2 is both necessary and sufficient to localize to MT plus ends and promote increased rates of MT polymerization, whereas the N-terminal region cannot bind to MT plus ends but can act in a dominant-negative capacity to reduce polymerization rates. Finally, we analyze mRNA expression patterns in Xenopus embryos for each TACC protein and observe neural enrichment of TACC3 expression compared with TACC1 and TACC2, which are also expressed in mesodermal tissues, including somites. Overall these data provide a novel assessment of all three TACC proteins as a family of +TIPs by highlighting the unique attributes of each, as well as their collective characteristics.

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.