Biology subjects

Winey, M.

Publications and source records attributed to Winey, M..

8 recordsLinked to original sources

bioRxiv2024

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.

cell biology↗

bioRxiv2021

Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from /{beta} tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of -tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a "molecular staple" stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

cell biology↗

bioRxiv2021

Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the {gamma}-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

cell biology↗

bioRxiv2020

Translational control of MPS1 links protein synthesis with the initiation of cell division and spindle pole body duplication in yeast

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the two poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the spindle pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their spindle pole body earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

cell biology↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

molecular biology↗

bioRxiv2019

Tetrahymena Poc5 is a transient basal body component that is important for basal body maturation

Basal bodies (BBs) are microtubule-based organelles that template and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation, and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised, and levels of BB overproduction are exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia.\n\nSUMMARY STATEMENTLoss of Tetrahymena thermophila Poc5 reveals an important role for this centrin-binding protein in basal body maturation, which also impacts basal body production and ciliogenesis.

cell biology↗

bioRxiv2019

Microtubule glycylation promotes basal body attachment to the cell cortex

Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BB) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. Using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincident with new BB assembly and are attached to the cell cortex. These BB-appendage microtubules are specifically marked with post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex for BB positioning, mutations that disrupt the cellular cortical cytoskeleton similarly disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.\n\nSUMMARY STATEMENTBasal bodies position motile cilia at the cell cortex. This study finds tubulin glycylation to promote BB-associated microtubule elongation and structural attachment of basal bodies to the cells cortical cytoskeleton.

cell biology↗

bioRxiv2018

Tetrahymena RIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged Microtubule Inner Proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical and imaging techniques we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins co-localize to Tetrahymena cilia and basal bodies, but assemble independently. Cryo-electron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4 and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.\n\nSUMMARYMicrotubule Inner Proteins (MIPs) bind to the luminal surface of highly stable microtubules. Combining cell biology and cryo-electron tomography, Stoddard et al. show that RIB72A and RIB72B are conserved MIPs in ciliary doublet microtubules and that they are important for proper ciliary motility.

cell biology↗