19 results on '"Kevin C. Slep"'
Search Results
2. Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker
- Author
-
Brian F. Saway, Rebecca C. Adikes, Kevin C. Slep, Ryan A. Hallett, and Brian Kuhlman
- Subjects
0301 basic medicine ,Microtubule dynamics ,Optogenetics ,Microtubules ,Tools ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Animals ,Cross linker ,Cytoskeleton ,Actin ,Research Articles ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,biology ,Schneider 2 cells ,Cell Biology ,biology.organism_classification ,Actins ,3. Good health ,Microtubule plus-end ,030104 developmental biology ,Cross-Linking Reagents ,Drosophila melanogaster ,Biophysics ,030217 neurology & neurosurgery ,Binding domain - Abstract
SxIP-iLID is a novel optogenetic tool designed to assess the temporal role of proteins on microtubule dynamics. The authors establish that optogenetic cross-linking of microtubule and actin networks decreases MT growth velocities and increases the cell area void of microtubules., We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes., Graphical Abstract
- Published
- 2018
3. TOG–tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation
- Author
-
Kevin C. Slep and Amy E. Byrnes
- Subjects
Models, Molecular ,0301 basic medicine ,Time Factors ,Mad2 ,Mitosis ,Spindle Apparatus ,Biology ,Transfection ,Microtubules ,Article ,Spindle pole body ,Cell Line ,Tubulin binding ,Microtubule polymerization ,Animals, Genetically Modified ,03 medical and health sciences ,Tubulin ,Microtubule ,Mitotic Index ,Animals ,Drosophila Proteins ,Protein Interaction Domains and Motifs ,Research Articles ,Microtubule nucleation ,Microtubule organizing center ,Cell Biology ,Cell biology ,Spindle apparatus ,Drosophila melanogaster ,030104 developmental biology ,Mad2 Proteins ,Mutation ,Protein Multimerization ,Microtubule-Associated Proteins ,Protein Binding - Abstract
Microtubule-associated proteins with arrays of TOG domains differentially regulate microtubule dynamics. Byrnes and Slep show that TOG arrays are polarized containing architecturally distinct TOG domains that bind either free or microtubule lattice-incorporated tubulin, which is essential for microtubule polymerization and mitotic spindle formation., XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family’s ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization. An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity. Requisite for this activity was a TOG5-specific N-terminal HEAT repeat that engaged microtubule lattice-incorporated tubulin. TOG5–microtubule binding maintained mitotic spindle formation as deleting or mutating TOG5 compromised spindle architecture and increased the mitotic index. Mad2 knockdown released the spindle assembly checkpoint triggered when TOG5–microtubule binding was compromised, indicating that TOG5 is essential for spindle function. Our results reveal a TOG5-specific role in mitotic fidelity and support our hypothesis that architecturally distinct TOGs arranged in a sequence-specific order underlie TOG array microtubule regulator activity.
- Published
- 2017
4. Structures of TOG1 and TOG2 From the Human Microtubule Dynamics Regulator CLASP1
- Author
-
Kevin C. Slep and Jonathan B. Leano
- Subjects
Models, Molecular ,Microtubule dynamics ,Polymers ,Protein Conformation ,Regulator ,Structural diversity ,Plasma protein binding ,Biochemistry ,Microtubules ,Conserved sequence ,Database and Informatics Methods ,Protein structure ,0302 clinical medicine ,Materials ,Cytoskeleton ,Conserved Sequence ,Polymerase ,0303 health sciences ,Crystallography ,Multidisciplinary ,biology ,Chemistry ,Physics ,Condensed Matter Physics ,Built Structures ,Recombinant Proteins ,Cell biology ,Macromolecules ,Cell Processes ,Physical Sciences ,Crystal Structure ,Engineering and Technology ,Medicine ,Cellular Structures and Organelles ,Sequence Analysis ,Microtubule-Associated Proteins ,Research Article ,Protein Binding ,Structural Engineering ,Bioinformatics ,Structural similarity ,Microtubule Polymerization ,Science ,Materials Science ,Sequence alignment ,Microtubule Dynamics ,Research and Analysis Methods ,Tubulin binding ,Structure-Activity Relationship ,03 medical and health sciences ,CLASP1 ,Tubulins ,Microtubule ,Electron Density ,Solid State Physics ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,030304 developmental biology ,Biology and Life Sciences ,Proteins ,Cell Biology ,Polymer Chemistry ,Cytoskeletal Proteins ,Tubulin ,Evolutionary biology ,biology.protein ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics.
- Published
- 2018
5. Crescerin uses a TOG domain array to regulate microtubules in the primary cilium
- Author
-
Kevin C. Slep, Cameron Champion Wood, Bob Goldstein, Alakananda Das, and Daniel J. Dickinson
- Subjects
Models, Molecular ,Protein family ,Microtubule-associated protein ,Regulator ,Microtubules ,Protein Structure, Secondary ,Conserved sequence ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Tubulin ,Microtubule ,Animals ,Amino Acid Sequence ,Cilia ,Caenorhabditis elegans ,Molecular Biology ,Conserved Sequence ,Cytoskeleton ,030304 developmental biology ,Neurons ,0303 health sciences ,biology ,Cilium ,Articles ,Cell Biology ,respiratory system ,biology.organism_classification ,Protein Structure, Tertiary ,Cell biology ,biology.protein ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Primary cilia are critical organelles involved in development, sensation, and signaling. Crescerin, a conserved protein family in ciliated and flagellated eukaryotes, uses a TOG domain array with tubulin polymerization activity to regulate cilia microtubules and facilitate proper cilia length, ultrastructure, and function., Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.
- Published
- 2015
6. The Secret of Centriole Length: Keep a LID on It
- Author
-
Kevin C. Slep
- Subjects
0301 basic medicine ,in vitro reconstitutions ,Centriole ,Protein domain ,Cell Cycle Proteins ,Biology ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,microtubules ,03 medical and health sciences ,Protein Domains ,Microtubule ,Organelle ,Animals ,Humans ,Basal body ,CPAP/SAS-4 ,Cell Cycle Protein ,Molecular Biology ,Centrioles ,X-ray crystallography ,human cells ,Cell Biology ,Cell biology ,Organelle structure ,030104 developmental biology ,Developmental Biology - Abstract
Summary Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and “caps” microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control., Highlights • CPAP's PN2-3 domain binds to an exposed site on β-tubulin at microtubule plus ends • CPAP tracks and caps microtubule plus ends in vitro • CPAP dampens microtubule growth in vitro • The capping function of CPAP limits centriolar microtubule growth in human cells, The mechanisms ensuring the extremely slow growth of centriolar microtubules remain elusive. Sharma, Aher, Dynes et al. demonstrate that human CPAP acts as a molecular lid that caps microtubule plus ends and dampens their elongation, thus contributing to centriole length control by ensuring slow processive assembly of centriolar microtubules.
- Published
- 2016
7. Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants
- Author
-
Kevin C. Slep, Elaine Fuchs, and Thomas J. Lane
- Subjects
0301 basic medicine ,Plasma protein binding ,Biology ,Bioinformatics ,Microtubules ,Article ,Focal adhesion ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Microtubule ,Cell polarity ,Humans ,EF Hand Motifs ,Molecular Biology ,Actin ,Binding Sites ,EF hand ,Microfilament Proteins ,Zinc ,030104 developmental biology ,HEK293 Cells ,MACF1 ,Mutation ,Biophysics ,Linker ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 A crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EFβ-scaffold with two bound Ca2+ ions that straddle an N-terminal α helix. The GAR domain has a unique α/β sandwich fold that coordinates Zn2+. While the EF1-EF2 domain is not sufficient for MT binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain's Zn2+-binding site, mediate MT binding.
- Published
- 2017
8. Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle
- Author
-
Nasser M. Rusan, Brandon Friedman, Julian Haase, Kerry Bloom, Amy E. Byrnes, Kevin C. Slep, Sarah K. Speed, Rebecca C. Adikes, Jaime C. Fox, Karen P. Haase, and Diana M. Cook
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Protein domain ,Saccharomyces cerevisiae ,Spindle Apparatus ,Microtubules ,Microtubule polymerization ,03 medical and health sciences ,Protein Domains ,Microtubule ,Tubulin ,Kinetochores ,Molecular Biology ,Cellular localization ,Cytoskeleton ,Coiled coil ,biology ,Kinetochore ,Cell Biology ,Articles ,3. Good health ,Cell biology ,Spindle apparatus ,030104 developmental biology ,biology.protein ,Protein Structural Elements ,Microtubule-Associated Proteins ,Protein Binding - Abstract
The yeast microtubule polymerase Stu2’s C-terminal domain is a 15-nm parallel, homodimeric coiled coil with two spatially distinct conserved regions. Determinants in these conserved regions optimally position Stu2 on the mitotic spindle to drive proper spindle structure and dynamics., XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain’s architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer’s N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.
- Published
- 2017
9. The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array
- Author
-
Kevin C. Slep, Joshua D. Currie, Jaime C. Fox, Amy E. Howard, and Stephen L. Rogers
- Subjects
Models, Molecular ,Microtubule-associated protein ,Xenopus ,Molecular Sequence Data ,Protein domain ,Spindle Apparatus ,Plasma protein binding ,Xenopus Proteins ,Crystallography, X-Ray ,Microtubules ,Microtubule polymerization ,03 medical and health sciences ,0302 clinical medicine ,Tubulin ,Microtubule ,Animals ,Drosophila Proteins ,Humans ,Amino Acid Sequence ,Molecular Biology ,Cytoskeleton ,030304 developmental biology ,0303 health sciences ,biology ,Articles ,Cell Biology ,Protein Structure, Tertiary ,3. Good health ,Spindle apparatus ,Cell biology ,biology.protein ,Drosophila ,Protein Multimerization ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery ,Drosophila Protein ,Protein Binding - Abstract
Structures of Drosophila Msps TOG4 and human ch-TOG TOG4 are presented. TOG4 departs from the other TOG structures and predicts novel α-tubulin engagement. Whereas TOG domains across the array have different tubulin-binding properties, cellular studies show that a fully functional TOG array is required for microtubule polymerase activity., XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization.
- Published
- 2014
10. A microtubule dynamics reconstitutional convention
- Author
-
Kevin C. Slep
- Subjects
0301 basic medicine ,biology ,Microtubule dynamics ,Microtubule-associated protein ,Polo kinase ,Cell Biology ,Spindle Apparatus ,Microtubules ,Models, Biological ,Spindle apparatus ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Tubulin ,Microtubule ,biology.protein ,Commentary ,Animals ,Humans ,Interphase ,Spotlight ,Mitosis ,Microtubule-Associated Proteins - Abstract
Moriwaki and Goshima identify the five molecular components that are necessary for recapitulation of all three phases of microtubule dynamics in vitro and show that Plk1 acts to shift microtubules to the mitotic mode., In vitro reconstitution is the fundamental test for identification of the core components of a biological process. In this issue, Moriwaki and Goshima (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201604118) reconstitute all phases of microtubule dynamics through the inclusion of five key regulators and demonstrate that Polo kinase activity shifts the system from an interphase mode into an enhanced mitotic mode.
- Published
- 2016
11. The Spectraplakin Short Stop Is an Actin–Microtubule Cross-Linker That Contributes to Organization of the Microtubule Network
- Author
-
Derek A, Applewhite, Kyle D, Grode, Darby, Keller, Alireza Dehghani, Zadeh, Alireza, Zadeh, Kevin C, Slep, and Stephen L, Rogers
- Subjects
0303 health sciences ,Articles ,Cell Biology ,Biology ,Microtubules ,Actins ,3. Good health ,Cell biology ,Cytoskeletal Proteins ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Morphogenesis ,Animals ,Drosophila ,Cross linker ,Molecular Biology ,Cytoskeleton ,030217 neurology & neurosurgery ,Actin ,Intracellular ,Protein Binding ,030304 developmental biology - Abstract
The dynamics of actin and microtubules are coordinated in many cellular processes, but little is known about molecules mediating cross-talk. We describe intracellular dynamics of Shot in a structure-function analysis of its role as a cross-linker. Shot interacts with microtubules two ways through EB1 and along microtubule lattices by the GAS2 domain., The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk.
- Published
- 2010
12. The role of TOG domains in microtubule plus end dynamics
- Author
-
Kevin C. Slep
- Subjects
Models, Molecular ,biology ,Protein Conformation ,Microtubule-associated protein ,Protein subunit ,Molecular Sequence Data ,Cell Cycle Proteins ,Sequence alignment ,Spindle Apparatus ,Xenopus Proteins ,Microtubules ,Biochemistry ,Cell biology ,Microtubule plus-end ,Xenopus laevis ,Tubulin ,Protein structure ,Microtubule ,biology.protein ,Animals ,Amino Acid Sequence ,Microtubule-Associated Proteins ,Sequence Alignment ,Peptide sequence - Abstract
The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain–tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.
- Published
- 2009
13. Structural Basis of Microtubule Plus End Tracking by XMAP215, CLIP-170, and EB1
- Author
-
Ronald D. Vale and Kevin C. Slep
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Time Factors ,Microtubule-associated protein ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Spindle Apparatus ,macromolecular substances ,Biology ,Crystallography, X-Ray ,Microtubules ,Models, Biological ,Article ,Tubulin binding ,Microtubule ,Animals ,Drosophila Proteins ,Humans ,Microtubule end ,Molecular Biology ,Microtubule nucleation ,Calcium-Binding Proteins ,Microfilament Proteins ,Biological Transport ,Cell Biology ,Neoplasm Proteins ,Protein Structure, Tertiary ,Microtubule plus-end ,Cell biology ,Drosophila melanogaster ,Tubulin ,Structural Homology, Protein ,Chromatography, Gel ,Microtubule Proteins ,biology.protein ,Dimerization ,Microtubule-Associated Proteins ,Microtubule plus-end binding ,HeLa Cells ,Protein Binding - Abstract
Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.
- Published
- 2007
14. Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in Drosophila Stem Cells
- Author
-
Brian J. Galletta, Karen M. Plevock, Kevin C. Slep, and Nasser M. Rusan
- Subjects
lcsh:Medicine ,Mitosis ,Spindle Apparatus ,Biology ,Crystallography, X-Ray ,Microtubules ,Spindle pole body ,Chromosomes ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Chromosome Segregation ,Animals ,Drosophila Proteins ,Clustered Regularly Interspaced Short Palindromic Repeats ,lcsh:Science ,030304 developmental biology ,Genetics ,Cell Nucleus ,Centrosome ,0303 health sciences ,Multidisciplinary ,Stem Cells ,lcsh:R ,Nuclear Proteins ,DNA ,Chromatin ,Cell biology ,Spindle apparatus ,Drosophila melanogaster ,lcsh:Q ,Spindle localization ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery ,Binding domain ,Research Article - Abstract
CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.
- Published
- 2015
15. Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface
- Author
-
Kevin C. Slep, Jaime C. Fox, and Amy E. Howard
- Subjects
Models, Molecular ,Microtubule-associated protein ,Molecular Sequence Data ,Gene Expression ,Mitosis ,Sequence alignment ,macromolecular substances ,Spindle Apparatus ,Plasma protein binding ,Crystallography, X-Ray ,Microtubules ,Biochemistry ,Protein Structure, Secondary ,Polymerization ,Tubulin binding ,Tubulin ,Microtubule ,Animals ,Drosophila Proteins ,Protein Isoforms ,Amino Acid Sequence ,Molecular Biology ,biology ,Protein Stability ,technology, industry, and agriculture ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Cell biology ,Crystallography ,Drosophila melanogaster ,Structural biology ,Protein Structure and Folding ,biology.protein ,Microtubule-Associated Proteins ,Sequence Alignment ,Protein Binding - Abstract
Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A-F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1-3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity.
- Published
- 2015
- Full Text
- View/download PDF
16. Structure of the Human Discs Large 1 PDZ2– Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
- Author
-
Kevin C. Slep
- Subjects
Macromolecular Assemblies ,Models, Molecular ,lcsh:Medicine ,Gene Expression ,PDZ Domains ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,Microtubules ,Discs Large Homolog 1 Protein ,0302 clinical medicine ,Molecular Cell Biology ,Cytoskeleton ,lcsh:Science ,0303 health sciences ,Multidisciplinary ,Stem Cells ,Recombinant Proteins ,Cell biology ,Cell Motility ,Structural Proteins ,Crystallization ,Research Article ,Protein Binding ,Protein Structure ,Adenomatous polyposis coli ,PDZ domain ,Adenomatous Polyposis Coli Protein ,Molecular Sequence Data ,Biophysics ,Biology ,Membrane-associated guanylate kinase ,03 medical and health sciences ,Microtubule ,Cell Adhesion ,Escherichia coli ,Animals ,Humans ,Amino Acid Sequence ,Protein Interactions ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,Binding Sites ,lcsh:R ,Proteins ,Membrane Proteins ,Epithelial Cells ,Cytoskeletal Proteins ,Membrane protein ,biology.protein ,lcsh:Q ,Peptides ,Sequence Alignment ,030217 neurology & neurosurgery ,Microtubule plus-end binding - Abstract
The membrane associated guanylate kinase (MAGUK) family member, human Discs Large 1 (hDlg1) uses a PDZ domain array to interact with the polarity determinant, the Adenomatous Polyposis Coli (APC) microtubule plus end binding protein. The hDLG1-APC complex mediates a dynamic attachment between microtubule plus ends and polarized cortical determinants in epithelial cells, stem cells, and neuronal synapses. Using its multi-domain architecture, hDlg1 both scaffolds and regulates the polarity factors it engages. Molecular details underlying the hDlg1-APC interaction and insight into how the hDlg1 PDZ array may cluster and regulate its binding factors remain to be determined. Here, I present the crystal structure of the hDlg1 PDZ2-APC complex and the molecular determinants that mediate APC binding. The hDlg1 PDZ2-APC complex also provides insight into potential modes of ligand-dependent PDZ domain clustering that may parallel Dlg scaffold regulatory mechanisms. The hDlg1 PDZ2-APC complex presented here represents a core biological complex that bridges polarized cortical determinants with the dynamic microtubule cytoskeleton.
- Published
- 2012
17. αβ-Tubulin and microtubule-binding assays
- Author
-
Jaime N, Campbell and Kevin C, Slep
- Subjects
Swine ,Tubulin ,Chromatography, Gel ,Animals ,Cattle ,Electrophoresis, Polyacrylamide Gel ,Microtubules ,Protein Binding - Abstract
Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, rescue, and severing. To confer these various functions, cytoskeletal regulators have highly tuned affinities for tubulin, recognizing the unpolymerized αβ-tubulin heterodimer, dynamic microtubule lattice, stabilized microtubule lattice, or a combination therein. The protocols presented here probe αβ-tubulin and microtubule binding using gel filtration and cosedimentation, respectively.
- Published
- 2011
18. Structural and mechanistic insights into microtubule end-binding proteins
- Author
-
Kevin C. Slep
- Subjects
Models, Molecular ,Protein Conformation ,fungi ,Molecular Sequence Data ,macromolecular substances ,Cell Biology ,Biology ,DNA-binding protein ,Microtubules ,In vitro ,Microtubule plus-end ,Cell biology ,Microtubule ,biology.protein ,Microtubule Proteins ,Tubulin polymerization ,Animals ,Humans ,Microtubule end ,Amino Acid Sequence ,Microtubule-Associated Proteins ,Polymerase ,Protein Binding - Abstract
Recent experiments reconstituting microtubule plus end tracking activity coupled with structural determination of microtubule plus end domains and plus end complexes are revealing the hierarchy, regulatory features, and potential mechanisms of plus end tracking proteins. Primary plus end tracking proteins include EB1 and XMAP215, while a host of secondary, EB1-dependent plus end proteins have been identified and characterized, including CLIP-170 and SKIP-motif proteins. Single molecule in vitro reconstitution assays show that XMAP215 is a processive polymerases that drives tubulin polymerization. Analysis of the EB1–microtubule interaction indicates EB1 actively promotes A-form microtubule lattice growth and rapidly exchanges with subsecond dwell times.
- Published
- 2009
19. Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end
- Author
-
Peter A. Kolodziej, Stephen L. Rogers, Ronald D. Vale, Kevin C. Slep, Sarah Elliott, and Hiroyuki Ohkura
- Subjects
Models, Molecular ,Protein family ,Cells ,1.1 Normal biological development and functioning ,Adenomatous Polyposis Coli Protein ,Amino Acid Motifs ,Molecular Sequence Data ,Plasma protein binding ,macromolecular substances ,Biology ,Microtubules ,Medical and Health Sciences ,Article ,03 medical and health sciences ,Microtubule ,Models ,Underpinning research ,Cell polarity ,Animals ,2.1 Biological and endogenous factors ,Amino Acid Sequence ,Aetiology ,Peptide sequence ,Research Articles ,Cells, Cultured ,030304 developmental biology ,Coiled coil ,0303 health sciences ,Cultured ,Binding Sites ,030302 biochemistry & molecular biology ,fungi ,Microfilament Proteins ,Molecular ,Cell Biology ,Biological Sciences ,Molecular biology ,Cell biology ,Microtubule plus-end ,MACF1 ,Mutagenesis ,Drosophila ,Generic health relevance ,Microtubule-Associated Proteins ,Protein Binding ,Developmental Biology - Abstract
EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.
- Published
- 2005
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.