A5071028767- Microtubule and mitosis dynamics
- Protist diversity and phylogeny
- Genetic and Kidney Cyst Diseases
- Photosynthetic Processes and Mechanisms
- Micro and Nano Robotics
- Algal biology and biofuel production
- Cellular transport and secretion
- Cellular Mechanics and Interactions
- Magnetic and Electromagnetic Effects
- 14-3-3 protein interactions
- Cell Adhesion Molecules Research
- RNA Research and Splicing
- Protein Structure and Dynamics
- Spaceflight effects on biology
- Lipid Membrane Structure and Behavior
- Genetics, Aging, and Longevity in Model Organisms
- Mechanisms of cancer metastasis
- Prenatal Screening and Diagnostics
- Genetics, Bioinformatics, and Biomedical Research
- Molecular Communication and Nanonetworks
- Antibiotic Use and Resistance
- Skin and Cellular Biology Research
- Wnt/β-catenin signaling in development and cancer
- Hemoglobin structure and function
- Antibiotics Pharmacokinetics and Efficacy
Emory University
1998-2024
Marquette University
2013-2023
University of California, Los Angeles
2007
Joint Genome Institute
2007
Institut de Biologie Physico-Chimique
2007
Sorbonne Université
2007
Centre National de la Recherche Scientifique
2007
Duke University
2007
University of Massachusetts Chan Medical School
2007
Pediatrics and Genetics
1994
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It model system for studying chloroplast-based photosynthesis, as well the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited common ancestor animals, but lost in plants. We sequenced ∼120-megabase nuclear genome performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated...
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-...
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...
Previous studies have shown that the polysialic acid (PSA) moiety associated with neural cell adhesion molecule (NCAM) has strong antiadhesive properties can influence a variety of cell-cell interactions.Based on size and structure this long linear homopolymer, we proposed activity PSA results from its physical properties.In present study, premise charge hydration underlie effects membrane vesicle aggregation is investigated through manipulation ionic strength.The establish are accentuated...
Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia sperm dysmotility. PCD characterized by clinical variability extensive genetic heterogeneity, associated different ultrastructural defects mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for diagnosis which not yet routine use. We developed targeted panel-based NGS...
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...
Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation controls bending, we cloned gene. encodes a novel protein calculated mass 111 kDa is predicted to form seven WD-repeats at C terminus. maps near BOP5 locus, bop5-1 contains point mutation resulting truncated lacking terminus, including seventh WD-repeat. cells display wild-type beat frequency but swim slower than cells,...
The unicellular alga Chlamydomonas can assemble two 10 μm flagella in 1 h from proteins synthesized the cell body. Targeting and transporting these to are simplified by preassembly of macromolecular complexes Radial spokes flagellar that partially assembled body before entering flagella. On axoneme, radial "T" shaped structures with a head five stalk 18 sediment together at 20S. In body, assembled; about half spoke (RSPs) form 12S complex. mutants lacking single RSP, smaller subassemblies...
The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding 1 (EB1) remains tip during flagellar shortening in absence intraflagellar transport (IFT), predominant system flagella. To investigate how accumulates tip, we used vivo imaging fluorescent protein-tagged (EB1-FP) Chlamydomonas reinhardtii. After photobleaching, signal recovered within minutes, indicating an exchange with unbleached entering flagella from cell body. moved independent...
Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse required to assemble remains largely unknown. Here, we show that radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated male infertility and reduced lung function. ARMC2 was highly enriched growing tagged spoke RSP3 co-migrated on anterograde trains. In contrast, cargo an adapter inner outer dynein arms moved independently...
We postulated that microcystin-sensitive protein phosphatases are integral components of the Chlamydomonas flagellar axoneme, positioned to regulate inner arm dynein activity. To test this, we took a direct biochemical approach. Microcystin-Sepharose affinity purification revealed prominent 35-kDa axonemal protein, predicted be catalytic subunit type-1 phosphatase (PP1c). cloned PP1c and produced specific polyclonal peptide antibodies. Based on western blot analysis, is anchored in axoneme....
Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted anchored to a unique site located proximal first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate positioning docking of complexes, we cloned characterized 140-kDa chain (IC140) complex. Sequence secondary analysis, with particular emphasis β-sheet organization, predicted IC140 contains seven WD repeats. Reexamination...
To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection motility mutants were generated by insertional mutagenesis. One such mutant, 5B10, lacks inner arm isoform known as I1 complex. This is located proximal to first radial spoke each 96-nm axoneme repeat an important target for regulation flagellar motility. Complementation tests reveal 5B10 represents locus, IDA7. Biochemical analyses confirm thatida7 axonemes lack at least...
ABSTRACT Genetic and morphological studies have revealed that the radial spokes regulate ciliary flagellar bending. Functional biochemical analysis discovery of calmodulin in suggest regulatory mechanism involves control axonemal protein phosphorylation calcium binding to spoke proteins. To identify potential proteins spoke, in-gel kinase assays were performed on isolated axonemes fractions. The results indicated 2 (RSP2) can bind ATP transfer phosphate vitro. RSP2 was cloned mapped PF24...
T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at spokehead–spokestalk juncture appear normal in length composition but twitch actively while cells jiggle without procession, resembling central pair (CP) mutant. HSP40− begin swimming upon electroporation with recombinant HSP40. Surprisingly, rescue doesn't require...
Our goal is to understand the assembly and regulation of flagellar dyneins, particularly Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel without notable structural domains. shares homology with murine lung adenoma susceptibility 1 (Las1) protein—a candidate tumor suppressor implicated in tumorigenesis. Multiple, independent biochemical assays determined that interacts both α- β-tubulin subunits within...
The radial spoke is a stable structural complex in the 9 + 2 axoneme for control of flagellar motility. However, spokes Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several proteins reduced differentially. To elucidate defective mechanism, we clone RSP16, prominent protein diminished axonemes. Unexpectedly, RSP16 novel HSP40 member DnaJ superfamily that assists chaperones various protein-folding-related processes. Importantly, uniquely excluded from 12S precursor...
Abstract Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial mechanochemical transducers that transmit signals from central pair apparatus to outer doublet microtubules local control of dynein activity. Recently, progress has been made in identifying individual components spoke, yet little is known about how spoke assembled or it performs signal transduction. Here we focus on protein 3...
LC8 is present in various molecular complexes. However, its role these complexes remains unclear. We discovered that although a subunit of the radial spoke (RS) complex Chlamydomonas flagella, it was undetectable RS precursor converted into mature at tip elongating axonemes. Interestingly, dimers bound tandem to N-terminal region phosphoprotein, protein 3 (RSP3), docks RSs enhanced binding RSP3 fragments purified Likewise, extracted from axonemes contained and putative spoke-docking...
A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on AH–RIIa interaction vitro. An RIIa or a similar Dpy-30 domain is also present numerous diverged molecules are implicated critical processes as diverse flagellar beating, membrane trafficking, histone methylation, stem cell differentiation, yet these remain poorly characterized....
We report the characterization of a profilin orthologue from Chlamydomonas reinhardtii. CrPRF, probably only isoform, is present in both cell body and flagella. Examination vegetative gametic cells by immunofluorescence microscopy using multiple fixation procedures also revealed enrichment CrPRF at anterior near base flagella fertilization tubule mating type plus gametes. Purified, recombinant binds to actin with Kd value ∼10–7 displaces nuclei live ‘nuclear displacement’ assay, consistent...
Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via network phosphoenzymes regulatory proteins. Consistently, Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes cAMP-dependent kinase (PKA) holoenzyme binding RIIa domain PKA subunit. However, highly docking is also found in N termini several AKAP-binding proteins unrelated well 24-kDa...
The radial spoke is a key element in transducer apparatus controlling the motility of eukaryotic cilia. transduction biomechanics long-standing question cilia biology. has three regions - head, bifurcated neck and stalk. Although stalk are asymmetric, twofold symmetry head remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate 3D structure whole at unprecedented resolution. We show region 15 Å (1.5 nm) resolution confirm symmetry....