Biology subjects

Gaertig, J.

Publications and source records attributed to Gaertig, J..

11 recordsLinked to original sources

bioRxiv2025

SPEF1 mediates assembly of the central pair microtubule complexes in cilia of Tetrahymena.

SPEF1 is a conserved protein that reinforces the seam of central microtubules within the distal segment of motile cilia. We show that in Tetrahymena, loss of SPEF1 destabilized the central pair throughout the cilium length, producing gaps in the microtubule lattice, loss of proximal microtubule portions, and loss of lateral projections. While both central microtubules were affected, C1 suffered more damage than C2 and the defects were already apparent during ciliary assembly. Tetrahymena cells lacking SPEF1 had fewer cilia, which contained an excessively dense ciliary matrix and abnormal densities on the doublet microtubules. Although SPEF1 loss visibly affected only the central microtubules, it localized to all stable microtubules in the cell, both ciliary and non-ciliary. Within cilia, SPEF1 was enriched near the distal tip, but was also present throughout the middle segment, where it was more concentrated along the central than the outer doublet microtubules. In live cilia, SPEF1 particles were mostly stationary and turned over slowly, some diffused but none moved by IFT. We propose that SPEF1 acts as a stabilizer of central microtubules in both the middle and distal segment and its activity contributes to central microtubule assembly.

cell biology↗

bioRxiv2024

Left-right cortical interactions drive intracellular pattern formation in the ciliate Tetrahymena

In ciliates, cortical organelles are positioned at precise locations along two polarity axes: anterior-posterior and circumferential (lateral). We explored the poorly understood mechanism of circumferential patterning, which generates left-right asymmetry. The model ciliate Tetrahymena has a single anteriorly-located oral apparatus. During cell division, a single new oral apparatus forms near the equator of the parental cell and along the longitude of the parental organelle. Cells homozygous for hypoangular 1 (hpo1) alleles, assemble multiple oral apparatuses positioned either to the left or right flanking the normal oral longitude. We identified HPO1 as a gene encoding an ARMC9-like protein. Hpo1 colocalizes with ciliary basal bodies, forming a bilateral concentration gradient with the high point on the cells right side and a sharp drop-off that marks the longitude at which oral development initiates on the ventral side. Hpo1 acts to exclude oral development from the cells right side. Hpo1 interacts with the Beige-Beach domain protein Bcd1, a cells left side-enriched factor, whose loss also confers formation of multiple oral apparatuses. A loss of both Hpo1 and Bcd1 is lethal and profoundly disrupts both positioning and organization of the forming oral apparatus (including its internal left-right polarity). We conclude that in ciliates, the circumferential/chiral patterning involves gradient-forming factors that are concentrated on either the cells right or left side and that the two sides of the cortex interact to create boundary effects that induce, position and shape developing cortical organelles.

cell biology↗

bioRxiv2024

The Janus A gene encodes a polo-kinase whose loss creates a dorsal/ventral intracellular homeosis in the ciliate, Tetrahymena.

Genetic studies on the protist, Tetrahymena thermophila provide a glimpse into the unexpectedly rich world of intracellular patterning that unfolds within the ciliate cell cortex. Ciliate pattern studies provide a useful counterpoint to animal models of pattern formation in that the unicellular model draws attention away from fields of cells (or nuclei) as the principal players in the metazoan pattern paradigm, focusing instead on fields of ciliated basal bodies serving as sources of positional information. In this study, we identify JANA, a Polo kinase of Tetrahymena, that serves as an important factor driving global, circumferential pattern. Loss of function of JanA results in global, mirror-duplication of ventral organelles on the dorsal surface: a kind of intracellular homeosis that has been named the janus phenotype. Gain of function (over-expression) reduces or even eliminates cortical organelles within the ventral hemi-cell. GFP-tagging reveals that JanA decorates basal bodies predominantly within the left-dorsal hemi-cell. These results led us to propose a model in which the default state of cortical patterning is a mirror-image assemblage of cortical organelles including oral apparatus, contractile vacuole pores and cytoproct. JanA normally suppresses organelle assembly in the dorsal hemi-cellular cortex, resulting in a simple, ventral assemblage of these organelles, a half-pattern as it were. PLK inhibitors produce a janus phenocopy, but reveal other unanticipated roles for PLK activities involving more local patterning events that control organelle dimensions and organization. We discuss results in light of metazoan studies in which PLK activity links cell cycle control to intracellular symmetry breaking.

developmental biology↗

bioRxiv2024

Structure of the ciliary tip central pair reveals the unique role of the microtubule-seam binding protein SPEF1

Motile cilia are unique organelles with the ability to autonomously move. Force generated by beating cilia propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet microtubules and a central pair (CP) with a distinct structure at the tip. In this study, we present a high-resolution structure of the CP in the ciliary tip of the ciliate Tetrahymena thermophila and identify several tip proteins that bind and form unique patterns on both microtubules of the tip CP. Two of those proteins that contain tubulin polymerization-promoting protein (TPPP)-like domains, TLP1 and TLP2, bind to high curvature regions of the microtubule. TLP2, which contains two TPPP-like domains, is an unusually long protein that wraps laterally around half a microtubule and forms the bridge between the two microtubules. Moreover, we found that the conserved protein SPEF1 binds to both microtubule seams. In vitro, human SPEF1 not only binds to the microtubule seam but also crosslinks two parallel microtubules. Single-molecule microtubule dynamics assays indicate that SPEF1 stabilizes microtubules in vitro. Together, these data show that the proteins in the tip CP maintain stable microtubule structure and probably play important roles in maintaining the integrity of the axoneme.

cell biology↗

bioRxiv2023

DomainFit: Identification of Protein Domains in cryo-EM maps at Intermediate Resolution using AlphaFold2-predicted Models

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of macromolecular complexes, enabling high-resolution structure determination. With the paradigm shift to in situ structural biology recently driven by the ground-breaking development of cryo-focused ion beam milling and cryo-electron tomography, there are an increasing number of structures at sub-nanometer resolution of complexes solved directly within their cellular environment. These cellular complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 [A] remains challenging because the side chains cannot be visualized reliably. Here, we present DomainFit, a program for automated domain-level protein identification from cryo-EM maps at resolutions lower than 4 [A]. By fitting domains from artificial intelligence-predicted models such as AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the proteins forming the density. Using DomainFit, we identified two microtubule inner proteins, one of them, a CCDC81 domain-containing protein, is exclusively localized in the proximal region of the doublet microtubule from the ciliate Tetrahymena thermophila. The flexibility and capability of DomainFit makes it a valuable tool for analyzing in situ structures.

biochemistry↗

bioRxiv2023

Effect of alpha-tubulin acetylation on the doublet microtubule structure

Acetylation of -tubulin at the lysine 40 residue (K40) by ATAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage-resistant. K40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that K40 acetylation exerts a small-scale effect on the doublet microtubule structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between K40 acetylation and phosphorylation in cilia.

cell biology↗

bioRxiv2023

Molecular architecture of the ciliary tip revealed by cryo-electron tomography

Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show the microtubules in the tip are highly cross-linked with each other and stabilised by luminal proteins, plugs and cap proteins at the plus ends. In the tip region, the central pair lacks the typical projections and twists significantly. By analysing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explain potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and inform about the mechanisms of ciliary assembly and length regulation.

cell biology↗

bioRxiv2020

Remodeling and activation mechanisms of outer arm dynein revealed by cryo-electron microscopy

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryoelectron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with molecular dynamics simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

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↗

bioRxiv2019

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. In Tetrahymena, a loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their total number per cell. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated the IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. A forward genetic screen identified a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. A loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. We propose that the total LF4/MOK activity per cilium is dependent on both its activation by an upstream CDK-related kinase and cilium length. Previous studies showed that the rate of assembly is high in growing cilia and decreases as cilia elongate to achieve the steady-state length. We propose that in a longer cilium, the IFT components, which travel from the base to the tip, are subjected to a higher dose of inhibition by the uniformly distributed LF4/MOK. Thus, in a feedback loop, LF4/MOK may translate cilium length into proportional inhibition of IFT, to balance the rates of assembly and disassembly at steady-state.\n\nAuthor summaryCilia are conserved organelles that generate motility and mediate vital sensory functions, including olfaction and vision. Cilia that are either too short or too long fail to generate proper forces or responses to extracellular signals. Several cilia-based diseases (ciliopathies) are associated with defects in cilia length. Here we use the multiciliated model protist Tetrahymena, to study a conserved protein kinase whose activity shortens cilia, LF4/MOK. We find that cells lacking an LF4/MOK kinase of Tetrahymena, LF4A, have excessively long, but also fewer cilia. We show that LF4A decreases the intraflagellar transport, a motility that shuttles ciliary precursors from the cilium base to the tip. Live imaging revealed that LF4A is distributed along cilium length and remains mostly immobile, likely due to its anchoring to ciliary microtubules. We proposed that in longer cilia, the intraflagellar transport machinery is exposed to a higher dose of inhibition by LF4A, which could decrease the rate of cilium assembly, to balance the rate of cilium disassembly in mature cilia that maintain stable length.

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↗