A5076154910- Microtubule and mitosis dynamics
- Protist diversity and phylogeny
- Genetic and Kidney Cyst Diseases
- Micro and Nano Robotics
- Photosynthetic Processes and Mechanisms
- Cellular transport and secretion
- Cellular Mechanics and Interactions
- 14-3-3 protein interactions
- Photoreceptor and optogenetics research
- Hemoglobin structure and function
- Blood properties and coagulation
- Erythrocyte Function and Pathophysiology
- Algal biology and biofuel production
- Cephalopods and Marine Biology
- Wnt/β-catenin signaling in development and cancer
- Magnetic and Electromagnetic Effects
- Reproductive Biology and Fertility
- Genetic Syndromes and Imprinting
- DNA and Nucleic Acid Chemistry
- Cardiomyopathy and Myosin Studies
- Congenital heart defects research
- Enzyme function and inhibition
- Transportation and Mobility Innovations
- Hippo pathway signaling and YAP/TAZ
- Caveolin-1 and cellular processes
Emory University
2015-2024
Albert Einstein College of Medicine
2014
Whitehead Institute for Biomedical Research
2008
Emory and Henry College
2008
State University of New York
1999
Rockefeller University
1990
University of Hawaii System
1977-1982
University of California, Berkeley
1976-1977
University of Hawaiʻi at Mānoa
1977
Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon the cells make contact surfaces, and flattening against is quite rapid. do not normally solid such sea urchin eggs, attach well so, amebas mammalian in culture. adhesion interpreted simply interaction between polyanionic cell polycationic layer adsorbed polylysine-treated can be exploited for a variety experimental manipulations. In preparation samples...
Axonemes of protozoan (Tetrahymena thermophila BIII) cilia, isolated by the dibucaine method, were treated briefly with trypsin after removal ciliary membranes treatment Triton X-100. After attachment to polylysine-coated surfaces, partially digested axonemes remained mainly intact cylinders. Such attached can be ATP, which induces microtubles sliding. ATP-treated preparations showed disrupted in doublets had telescoped out original These could captured place for electron microscopy critical...
The motility of demembranated sea urchin sperm flagella and that embryo cilia reactivated with 0.1 mM ATP are completely inhibited by 4 micron 0.5 vanadium(V) [V(V), in vanadate], respectively. Mg2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3)of the latent form dynein 1 is 50% 0.5-1 V(V), while Ca2+-activated much less sensitive. inhibition flagellar beat frequency V(V) appears not to be competitive ATP. In agreement other reports, (Na,K)-ATPase shows a slow onset presence...
The molecular composition and organization of the row axonemal inner dynein arms were investigated by biochemical electron microscopic analyses Chlamydomonas wild-type mutant axonemes. Three arm structures could be distinguished on basis their position in axoneme as determined analysis pf30 pf23 mutants. three repeat every 96 nm are referred to here I1, I2, I3. I1 is proximal radial spoke S1, whereas I2 I3 distal spokes S1 S2, respectively. lacks both but has a normal Each six heavy chains...
The radial spoke is a ubiquitous component of '9+2' cilia and flagella, plays an essential role in the control dynein arm activity by relaying signals from central pair microtubules to arms. Chlamydomonas reinhardtii contains at least 23 proteins, only 8 which have been characterized molecular level. Here, we use mass spectrometry identify 10 additional proteins. Many newly identified proteins stalk are predicted contain domains associated with signal transduction, including Ca2+-, AKAP-...
The regulation of microtubule sliding in flagellar axonemes was studied with the use Chlamydomonas mutants and vitro assays. Microtubule velocities were diminished from mutant cells missing radial spoke structures but could be restored upon reconstitution dynein wild-type spokes. These experiments demonstrate that spokes activate dynein's activity.
The nexin-dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into axoneme. At least 10 subunits associate with form discrete distinct from other axonemal substructures. Transformation drc4 mutants epitope-tagged rescues motility defects restores missing DRC associated inner-arm dyneins. Four new contain calcium-signaling motifs and/or AAA domains...
The motile cilium is a mechanical wonder, cellular nanomachine that produces high-speed beat based on cycle of bends move along an axoneme made 9+2 microtubules. molecular motors, dyneins, power the ciliary beat. dyneins are compacted into inner and outer dynein arms, whose activity highly regulated to produce microtubule sliding axonemal bending. switch point hypothesis was developed long ago account for how in presence radial spoke–central pair interactions causes Since then, new genetic,...
Genetic and in vitro analyses have revealed that radial spokes play a crucial role regulation of ciliary flagellar motility, including control waveform. However, the mechanisms are not understood. Here, we developed novel procedure to isolate intact as step toward understanding mechanism by which these complexes regulate dynein activity. The isolated sediment 20S size shape spokes. Extracted rescue spoke structure when reconstituted with axonemes derived from mutant pf14. Isolated composed...
One of the challenges in understanding ciliary and flagellar motility is determining mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated sliding, Chlamydomonas flagella, regulated by phosphorylation. However, regulatory proteins remain unknown. Here we identify 138-kD intermediate chain inner arm dynein I1 as critical phosphoprotein required for regulation motility. This conclusion founded on results three different experimental approaches....
Outer-arm dynein from the sperm of sea urchin S. purpuratus was adsorbed to mica flakes and visualized by quick-freeze, deep-etch technique. Replicas reveal particles comprised two globular heads joined irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) other spherical (14.5-nm diam). The are decorated a complex bead-like subunits. same two-headed protein found in 21S dynein-1 fraction sucrose gradients. beta-heavy chain/intermediate...
Structural studies of stationary principal bends and definitive patterns spontaneous microtubule sliding disruption permitted description the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus were demembranated a buffer containing Triton X-100 EGTA. Subsequent resuspension reactivation 0.4 mM CaCl2 1.0 MgATP2- resulted quiescent, rather than motile, cells each axoneme took on an extreme, basal bend 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously...
Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 mia2, which display slow swimming low flagellar beat frequency. found that the MIA1 MIA2 genes encode conserved coiled-coil proteins, FAP100 FAP73, respectively, form modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography mia mutant axonemes revealed MIA was located...
Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) regulates axonemal motility dynein activity. However, mechanism for anchoring PKA in is unknown. Here we test hypothesis an A-kinase (AKAP). By performing RII blot overlays on mutants defective specific structures, two AKAPs been identified: 240-kD AKAP associated with central pair apparatus, 97-kD located radial spoke stalk. Based...
Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with pair microtubules and reduce beat frequency, but do not prevent changes activity either photophobic responses or phototactic accumulation of live cells. Comparison cpc1 pf6 axonemes shows that affects row projections along C1 distinct from those missing pf6, thin fibers form an arc between the two...
Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas the 138-kDa intermediate chain (IC138) of inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) physically anchored flagellar axoneme regulates IC138 phosphorylation activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued...
Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding Chlamydomonas flagella. However, the molecular mechanism by dynein activity is regulated unknown. We describe results from three different vitro approaches to test hypothesis that an protein kinase inhibits spoke-deficient axonemes First, velocity of mutants (pf14, pf17) was increased wild-type level after treatment with inhibitors HA-1004 or H-7 specific...
Our goal was to assess the microtubule translocating ability of individual ATPase subunits outer arm dynein. Solubilized dynein from sea urchin sperm (Stronglocentrotus purpuratus) dissociated into by low ionic strength buffer and fractionated zonal centrifugation. Fractions were assessed an in vitro functional assay wherein microtubules move across a glass surface which isolated fractions had been absorbed. Microtubule gliding activity coincident with 12-S beta-heavy chain-intermediate...