Biology subjects

Joachimiak, E.

Publications and source records attributed to Joachimiak, E..

6 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

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

Heterogeneity of radial spokes structural components and associated enzymes in Tetrahymena cilia

Radial spokes, RS1-RS2-RS3, are T-shaped, multiprotein complexes that transmit regulatory signals from central apparatus to outer doublet dyneins. RSs, especially RS3, differ in morphology, protein composition, and RS base-docked IDAs. Spokes defects alter cilia beating frequency, waveform, and amplitude leading, in humans, to primary ciliary dyskinesia and infertility. In contrast to RS1 and RS2, the protein composition of RS3 is partly resolved. Moreover, the role of particular spokes is unclear. Ciliate Tetrahymena thermophila has three Rsp3 paralogs and two or three paralogs of some other RSPs. Using multiple complementary approaches, we showed that Tetrahymena forms RS1 and RS2 subtypes having core composed of various Rsp3 paralogs and one type of Rsp3-less RS3. We elucidated proteomes of RS subtypes and identified novel RS-associated proteins, including enzymatic proteins involved in local regulation of the ADP/ATP levels and protein phosphorylation, whose presence further diversifies RSs properties and likely functions. In briefRadial spokes differ in their protein composition and architecture. Studies in a ciliate Tetrahymena revealed Rsp3 paralogs-dependent RS1 and RS2 subtypes, Rsp3-less RS3, and diversity of the RSP3 mutants phenotype. Known RS components and newly identified structural and enzymatic proteins were assigned to particular RSs.

cell biology↗

bioRxiv2022

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 38 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

biophysics↗

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↗