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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Fap206 Is A Microtubule-Docking Adapter For Ciliary Radial Spoke 2 And Dynein C, Krishna K. Vasudevan, Kangkang Song, Lea M. Alford, Winfield Sale, Erin E. Dymek, Elizabeth F. Smith, Todd Hennessey, Ewa Joachimiak, Paulina Urbanska, Dorota Wloga, William Dentler, Daniela Nicastro, Jacek Gaertig Dec 2014

Fap206 Is A Microtubule-Docking Adapter For Ciliary Radial Spoke 2 And Dynein C, Krishna K. Vasudevan, Kangkang Song, Lea M. Alford, Winfield Sale, Erin E. Dymek, Elizabeth F. Smith, Todd Hennessey, Ewa Joachimiak, Paulina Urbanska, Dorota Wloga, William Dentler, Daniela Nicastro, Jacek Gaertig

Open Dartmouth: Faculty Open Access Scholarship

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but ...


Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic Jan 2012

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Open Dartmouth: Faculty Open Access Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to ...


Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs Sep 2011

Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs

Open Dartmouth: Faculty Open Access Scholarship

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.


The Pcdp1 Complex Coordinates The Activity Of Dynein Isoforms To Produce Wild-Type Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith Sep 2011

The Pcdp1 Complex Coordinates The Activity Of Dynein Isoforms To Produce Wild-Type Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith

Open Dartmouth: Faculty Open Access Scholarship

Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities ...


The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith May 2011

The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith

Open Dartmouth: Faculty Open Access Scholarship

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and ...


A Kinesin Motor In A Force-Producing Conformation, Elisabeth Heuston, C. Eric Bronner, F Jon Kull, Sharyn A. Endow Jul 2010

A Kinesin Motor In A Force-Producing Conformation, Elisabeth Heuston, C. Eric Bronner, F Jon Kull, Sharyn A. Endow

Open Dartmouth: Faculty Open Access Scholarship

Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states.


Examining The Link Between Chromosomal Instability And Aneuploidy In Human Cells, Sarah L. Thompson, Duane A. Compton Jan 2008

Examining The Link Between Chromosomal Instability And Aneuploidy In Human Cells, Sarah L. Thompson, Duane A. Compton

Open Dartmouth: Faculty Open Access Scholarship

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule–chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise ...


A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith Nov 2007

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Open Dartmouth: Faculty Open Access Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which ...


The Kini Kinesin Kif2a Is Required For Bipolar Spindle Assembly Through A Functional Relationship With Mcak, Neil J. Ganem, Duane A. Compton Aug 2004

The Kini Kinesin Kif2a Is Required For Bipolar Spindle Assembly Through A Functional Relationship With Mcak, Neil J. Ganem, Duane A. Compton

Open Dartmouth: Faculty Open Access Scholarship

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for ...


Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor Mar 2003

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Open Dartmouth: Faculty Open Access Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established ...