Ewa Joachimiak - Academia.edu (original) (raw)

Papers by Ewa Joachimiak

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory function... more 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...

Seminars in Cell & Developmental Biology

Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization ... more Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium, to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms.

Acta Biochimica Polonica, 2010

Journal of Cell Biology, 2020

Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern ... more Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern is propagated during “tandem duplication,” a cell division that remodels the parental cell into two daughter cells. A key step is the formation of the division boundary along the cell’s equator. In Tetrahymena thermophila, the cdaA alleles prevent the formation of the division boundary. We find that the CDAA gene encodes a cyclin E that accumulates in the posterior cell half, concurrently with accumulation of CdaI, a Hippo/Mst kinase, in the anterior cell half. The division boundary forms between the margins of expression of CdaI and CdaA, which exclude each other from their own cortical domains. The activities of CdaA and CdaI must be balanced to initiate the division boundary and to position it along the cell’s equator. CdaA and CdaI cooperate to position organelles near the new cell ends. Our data point to an intracellular positioning mechanism involving antagonistic Hippo signaling a...

European Journal of Protistology, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Journal of Eukaryotic Microbiology, 2005

Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation ... more Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation of Tetrahymena by formation of specific structures during these processes. In cell cycle it appears at the anterior poles of future proter and opisth cells. By 2-D electrophoresis, we found ...

European Journal of Protistology, 2013

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, seve... more In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycleregulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.

Developmental Biology, 1999

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized cil... more In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.

Journal of Cellular Physiology, 2018

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorl... more The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B-or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.

Journal of Cellular Physiology, 2013

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chape... more Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Insects

The molecular oscillator is the core of the biological clock and is formed by genes and proteins ... more The molecular oscillator is the core of the biological clock and is formed by genes and proteins whose cyclic expression is regulated in the transcriptional-translational feedback loops (TTFLs). Proteins of the TTFLs are regulators of both their own and executive genes involved in the control of many processes in insects (e.g., rhythmic metabolism of xenobiotics, including insecticides). We disrupted the clock operation in S. littoralis larvae by injecting the dsRNA of clock genes into their body cavity and culturing the larvae under continuous light. As a result, the daily susceptibility of larvae to insecticides was abolished and the susceptibility itself increased (in most cases). In the fat body, midgut, and Malpighian tubules (the main organs metabolizing xenobiotics) of the larvae treated with injected-dsRNA, the daily activity profiles of enzymes involved in detoxification—cytochrome P450 monooxygenases, Glutathione-S-transferase, and esterase—have changed significantly. The ...

PLOS Genetics, 2021

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein com... more Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

International Journal of Molecular Sciences, 2022

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile ci... more Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000–20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas, the parasitic protist Trypanosoma, and free-living ciliates, Tetrahymena and Paramecium, the invertebrate Schmidtea, and vertebrates such as zebrafish, Xenopus, and mouse. Establishing such evolutionarily distant experimental models with different levels of cell...

International Journal of Molecular Sciences, 2021

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enab... more Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.

Scientific Reports, 2021

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The... more Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, ...

Molecules, 2020

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are invo... more Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the conse...

Kosmos, 2018

Rzęski pierwotne, struktury zbudowane na bazie cytoszkieletu mikrotubularnego, występują na powie... more Rzęski pierwotne, struktury zbudowane na bazie cytoszkieletu mikrotubularnego, występują na powierzchni niemal wszystkich komórek ssaczych. Dzięki licznym receptorom błonowym, rzęski pierwotne pośredniczą w odbieraniu i przekazywaniu bodźców ze środowiska do wnętrza komórki, i tym samym odgrywają niezwykle ważną rolę w prawidłowym rozwoju i funkcjonowaniu większości tkanek i narządów. Tworzenie rzęski (ciliogeneza) to złożony, wieloetapowy i wielopoziomowo regulowany proces ściśle związany z cyklem komórkowym. Mutacje w genach kodujących białka strukturalne lub odpowiedzialne za prawidłowe funkcjonowanie rzęsek, jak również, regulujące przebieg ciliogenezy są przyczyną ich dysfunkcji, prowadzącej w efekcie do wielonarządowych chorób zwanych ciliopatiami.

Kosmos, 2018

Rzęski są strukturami zachowanymi w toku ewolucji, występującymi u większości Eukaryota. Ze wzglę... more Rzęski są strukturami zachowanymi w toku ewolucji, występującymi u większości Eukaryota. Ze względu na strukturę i pełnione funkcje wyróżnia się dwa typy rzęsek: nieruchome rzęski pierwotne, tworzone w fazie spoczynkowej cyklu komórkowego oraz rzęski ruchome. Rzęski pierwotne są odpowiedzialne za odbieranie i przekazywanie sygnałów ze środowiska do wnętrza komórki, natomiast rzęski ruchome umożliwiają ruch pojedynczych komórek, a w organizmach wielokomórkowych, w tym u człowieka, przemieszczanie wydzielin lub drobin wzdłuż powierzchni komórek nabłonka wyścielającego m.in. drogi oddechowe, jajowód i komory mózgowia. Szkielet obu typów rzęsek, tzw. aksomena, zbudowany jest z dziewięciu obwodowych par mikrotubul. Rzęski ruchome mają dodatkowo dwie mikrotubule centralne, które wraz z przyłączonymi do nich kompleksami białkowymi tworzą kompleks pary centralnej, oraz makrokompleksy białek przyłączone do mikrotubul obwodowych. Makrokompleksy te są rozmieszczone periodycznie wzdłuż mikrotub...

Kosmos, 2018

Ciałko podstawowe i centriola to struktury homologiczne, których zrąb stanowi dziewięć mikrotubul... more Ciałko podstawowe i centriola to struktury homologiczne, których zrąb stanowi dziewięć mikrotubularnych tripletów. Mikrotubulom ciałka podstawowego/centrioli towarzyszą liczne struktury mikrotubularne i niemikrotubularne. Ich obecność nie tylko powoduje polaryzację ciałka podstawowego i centrioli, lecz także umożliwia ich prawidłowe funkcjonowanie. Przypuszcza się, że ciałka podstawowe występowały już u ostatniego wspólnego przodka eukariontów, tzw. LECA, a ich budowa i funkcja okazały się tak wydajne, że nie zmieniły się znacząco w toku ewolucji. Ciałka podstawowe i centriole odgrywają istotną rolę w komórce, a zaburzenia ich liczby, struktury lub lokalizacji obserwuje się m.in. w licznych nowotworach, chorobach układu nerwowego czy złożonych zespołach wieloobjawowych zwanych ciliopatiami.

Kosmos, 2018

Wnętrze rzęski nie jest oddzielone od cytoplazmy błoną biologiczną, a mimo to ma unikatowy skład.... more Wnętrze rzęski nie jest oddzielone od cytoplazmy błoną biologiczną, a mimo to ma unikatowy skład. Jest to możliwe dzięki działaniu zlokalizowanej u podstawy rzęski tzw. bariery rzęskowej. W skład tej struktury wchodzi dystalna część ciałka podstawowego, proksymalna część rzęski, umiejscowione na nich włókna przejściowe i łączniki Y, a także fragmenty przylegającej do nich błony komórkowej i rzęskowej. Tak złożona budowa umożliwia z jednej strony zatrzymanie u podstawy rzęski białek niepożądanych, a z drugiej, ułatwienie transportu do wnętrza rzęski elementów niezbędnych do jej budowy i funkcjonowania.

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory function... more 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...

Seminars in Cell & Developmental Biology

Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization ... more Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium, to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms.

Acta Biochimica Polonica, 2010

Journal of Cell Biology, 2020

Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern ... more Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern is propagated during “tandem duplication,” a cell division that remodels the parental cell into two daughter cells. A key step is the formation of the division boundary along the cell’s equator. In Tetrahymena thermophila, the cdaA alleles prevent the formation of the division boundary. We find that the CDAA gene encodes a cyclin E that accumulates in the posterior cell half, concurrently with accumulation of CdaI, a Hippo/Mst kinase, in the anterior cell half. The division boundary forms between the margins of expression of CdaI and CdaA, which exclude each other from their own cortical domains. The activities of CdaA and CdaI must be balanced to initiate the division boundary and to position it along the cell’s equator. CdaA and CdaI cooperate to position organelles near the new cell ends. Our data point to an intracellular positioning mechanism involving antagonistic Hippo signaling a...

European Journal of Protistology, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Journal of Eukaryotic Microbiology, 2005

Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation ... more Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation of Tetrahymena by formation of specific structures during these processes. In cell cycle it appears at the anterior poles of future proter and opisth cells. By 2-D electrophoresis, we found ...

European Journal of Protistology, 2013

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, seve... more In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycleregulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.

Developmental Biology, 1999

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized cil... more In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.

Journal of Cellular Physiology, 2018

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorl... more The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B-or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.

Journal of Cellular Physiology, 2013

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chape... more Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Insects

The molecular oscillator is the core of the biological clock and is formed by genes and proteins ... more The molecular oscillator is the core of the biological clock and is formed by genes and proteins whose cyclic expression is regulated in the transcriptional-translational feedback loops (TTFLs). Proteins of the TTFLs are regulators of both their own and executive genes involved in the control of many processes in insects (e.g., rhythmic metabolism of xenobiotics, including insecticides). We disrupted the clock operation in S. littoralis larvae by injecting the dsRNA of clock genes into their body cavity and culturing the larvae under continuous light. As a result, the daily susceptibility of larvae to insecticides was abolished and the susceptibility itself increased (in most cases). In the fat body, midgut, and Malpighian tubules (the main organs metabolizing xenobiotics) of the larvae treated with injected-dsRNA, the daily activity profiles of enzymes involved in detoxification—cytochrome P450 monooxygenases, Glutathione-S-transferase, and esterase—have changed significantly. The ...

PLOS Genetics, 2021

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein com... more Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

International Journal of Molecular Sciences, 2022

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile ci... more Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000–20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas, the parasitic protist Trypanosoma, and free-living ciliates, Tetrahymena and Paramecium, the invertebrate Schmidtea, and vertebrates such as zebrafish, Xenopus, and mouse. Establishing such evolutionarily distant experimental models with different levels of cell...

International Journal of Molecular Sciences, 2021

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enab... more Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.

Scientific Reports, 2021

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The... more Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, ...

Molecules, 2020

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are invo... more Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the conse...

Kosmos, 2018

Rzęski pierwotne, struktury zbudowane na bazie cytoszkieletu mikrotubularnego, występują na powie... more Rzęski pierwotne, struktury zbudowane na bazie cytoszkieletu mikrotubularnego, występują na powierzchni niemal wszystkich komórek ssaczych. Dzięki licznym receptorom błonowym, rzęski pierwotne pośredniczą w odbieraniu i przekazywaniu bodźców ze środowiska do wnętrza komórki, i tym samym odgrywają niezwykle ważną rolę w prawidłowym rozwoju i funkcjonowaniu większości tkanek i narządów. Tworzenie rzęski (ciliogeneza) to złożony, wieloetapowy i wielopoziomowo regulowany proces ściśle związany z cyklem komórkowym. Mutacje w genach kodujących białka strukturalne lub odpowiedzialne za prawidłowe funkcjonowanie rzęsek, jak również, regulujące przebieg ciliogenezy są przyczyną ich dysfunkcji, prowadzącej w efekcie do wielonarządowych chorób zwanych ciliopatiami.

Kosmos, 2018

Rzęski są strukturami zachowanymi w toku ewolucji, występującymi u większości Eukaryota. Ze wzglę... more Rzęski są strukturami zachowanymi w toku ewolucji, występującymi u większości Eukaryota. Ze względu na strukturę i pełnione funkcje wyróżnia się dwa typy rzęsek: nieruchome rzęski pierwotne, tworzone w fazie spoczynkowej cyklu komórkowego oraz rzęski ruchome. Rzęski pierwotne są odpowiedzialne za odbieranie i przekazywanie sygnałów ze środowiska do wnętrza komórki, natomiast rzęski ruchome umożliwiają ruch pojedynczych komórek, a w organizmach wielokomórkowych, w tym u człowieka, przemieszczanie wydzielin lub drobin wzdłuż powierzchni komórek nabłonka wyścielającego m.in. drogi oddechowe, jajowód i komory mózgowia. Szkielet obu typów rzęsek, tzw. aksomena, zbudowany jest z dziewięciu obwodowych par mikrotubul. Rzęski ruchome mają dodatkowo dwie mikrotubule centralne, które wraz z przyłączonymi do nich kompleksami białkowymi tworzą kompleks pary centralnej, oraz makrokompleksy białek przyłączone do mikrotubul obwodowych. Makrokompleksy te są rozmieszczone periodycznie wzdłuż mikrotub...

Kosmos, 2018

Ciałko podstawowe i centriola to struktury homologiczne, których zrąb stanowi dziewięć mikrotubul... more Ciałko podstawowe i centriola to struktury homologiczne, których zrąb stanowi dziewięć mikrotubularnych tripletów. Mikrotubulom ciałka podstawowego/centrioli towarzyszą liczne struktury mikrotubularne i niemikrotubularne. Ich obecność nie tylko powoduje polaryzację ciałka podstawowego i centrioli, lecz także umożliwia ich prawidłowe funkcjonowanie. Przypuszcza się, że ciałka podstawowe występowały już u ostatniego wspólnego przodka eukariontów, tzw. LECA, a ich budowa i funkcja okazały się tak wydajne, że nie zmieniły się znacząco w toku ewolucji. Ciałka podstawowe i centriole odgrywają istotną rolę w komórce, a zaburzenia ich liczby, struktury lub lokalizacji obserwuje się m.in. w licznych nowotworach, chorobach układu nerwowego czy złożonych zespołach wieloobjawowych zwanych ciliopatiami.

Kosmos, 2018

Wnętrze rzęski nie jest oddzielone od cytoplazmy błoną biologiczną, a mimo to ma unikatowy skład.... more Wnętrze rzęski nie jest oddzielone od cytoplazmy błoną biologiczną, a mimo to ma unikatowy skład. Jest to możliwe dzięki działaniu zlokalizowanej u podstawy rzęski tzw. bariery rzęskowej. W skład tej struktury wchodzi dystalna część ciałka podstawowego, proksymalna część rzęski, umiejscowione na nich włókna przejściowe i łączniki Y, a także fragmenty przylegającej do nich błony komórkowej i rzęskowej. Tak złożona budowa umożliwia z jednej strony zatrzymanie u podstawy rzęski białek niepożądanych, a z drugiej, ułatwienie transportu do wnętrza rzęski elementów niezbędnych do jej budowy i funkcjonowania.