Composition and function of the C1b/C1f region in the ciliary central apparatus

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, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants

FAP206 is a Microtubule-Docking Adapter for Ciliary Radial Spoke 2 and Dynein c

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule

Structure of basal body and centriole

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.Basal body and centriole are homologous structures, build of nine triplet microtubules. The basal body/centriole microtubular scaffold is accompanied by numerous structures both microtubular and non-microtubular, which not only cause basal body/centriole polarization but also allow its proper functioning. It is assumed that basal bodies were present in last common eukaryotic ancestor, so-called LECA, and their structure and function appeared such efficient, that they did not change significantly in evolution. Basal bodies and centrioles play an important role in the cell and the abnormalities in their number, structure or location are observed in numerous cancers, neuropathies and ciliopathies

On the basal body and cilium border - ciliary gate

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.The intraciliary space is not separated from the cell cytoplasm by a membrane, but still it has a unique composition. It is possible due to the existence of so-called ciliary gate localized at the ciliary base. This structure is composed of the distal part of basal body, proximal portion of cilium, transition fibers and Y-links and adjacent part of the cell and ciliary membrane. This complex structure, on one hand retains the unwanted proteins at the ciliary base and, on the other hand, facilitates the intraciliary transport of cargos required for cilia formation and function

Biogenesis of centrioles

Centriole są mikrotubularnymi strukturami wchodzącymi w skład centrosomu, występującymi u zwierząt. Struktury te pełnią istotne funkcje zarówno podczas interfazy i mitozy komórek proliferujących, jak i w zróżnicowanych komórkach, włączając nabłonki orzęsione. Liczba centrioli w komórce jest ściśle kontrolowana, gdyż ich nadmiar lub niedobór prowadzi do zaburzeń podziału komórkowego lub funkcjonowania komórek. Centriole powstają w procesie zwanym biogenezą lub duplikacją. W komórkach proliferujących centriole są powielane jeden raz, podczas gdy w komórkach nabłonków orzęsionych dochodzi do namnożenia większej ich liczby, co związane jest z odmiennym mechanizmem powielania tych organelli. W procesie tzw. "klasycznej" duplikacji, zwanej też "centriolarną", która ma miejsce w komórkach dzielących się, dochodzi do wytworzenia dwóch nowych centrioli. Z kolei podczas tzw. duplikacji "deuterosomalnej", w różnicujących komórkach nabłonków orzęsionych wytwarzanych jest nawet kilkaset centrioli.Centrioles are microtubule-based organelles that in animal cells are a part of the centrosome. These structures play an important role during both interphase and mitosis in proliferating cells, as well as in differentiated cells, including ciliation of epithelial cells. The number of centrioles is strictly regulated as their excess or reduction leads to abnormalities in cell division or cell functions. Centrioles are formed in a process called biogenesis or duplication. In proliferating cells, centrioles are duplicated only once in each cell cycle, while in epithelial cells before ciliation, numerous centrioles are assembled, suggesting different mechanisms of duplication. During the so-called "classic" or "centriolar" duplication, in proliferating cells two new centrioles are formed before mitosis. By contrast, during differentiating into ciliated epithelial cells even few hundreds of new centrioles are assembled (so-called "deuterosomal" biogenesis)

Tubulin Post-Translational Modifications and Microtubule Dynamics

Microtubules are hollow tube-like polymeric structures composed of α,β-tubulin heterodimers. They play an important role in numerous cellular processes, including intracellular transport, cell motility and segregation of the chromosomes during cell division. Moreover, microtubule doublets or triplets form a scaffold of a cilium, centriole and basal body, respectively. To perform such diverse functions microtubules have to differ in their properties. Post-translational modifications are one of the factors that affect the properties of the tubulin polymer. Here we focus on the direct and indirect effects of post-translational modifications of tubulin on microtubule dynamics

Biogenesis of the primary cilium

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.Cilia are highly specialized, microtubule-based protrusions, extended on cell surface in almost all mammalian cell types. They function as cell antennae that receive and transmit signals from the environment to the cell body. Cilia formation, so-called ciliogenesis is strictly controlled at multiple levels by a number of proteins, and correlated with the cell cycle progression. Cilia dysfunctions cause a wide range of human diseases, called ciliopathies. Moreover, ciliary defects may lead to obesity and cancer. In this article, we summarize current knowledge concerning cilia function and structure, regulation of ciliogenesis, and the most important signaling pathways and diseases affected by cilia dysfunction

Yolk protein is expressed in the insect testis and interacts with sperm

<p>Abstract</p> <p>Background</p> <p>Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, <it>Spodoptera littoralis</it>, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation.</p> <p>Results</p> <p>We collected seminal fluid proteins from males before and after daily sperm release. These samples were separated by 2-D gel electrophoresis, and gels were treated with a glycoprotein-detecting probe. We observed a group of abundant glycoproteins in the sample collected after sperm release, which was absent in the sample collected before sperm release. Sequencing of these glycoproteins by mass spectroscopy revealed peptides bearing homology with components of yolk, which is known to accumulate in developing oocytes. This unexpected result was confirmed by Western blotting demonstrating that seminal fluid contains protein immunoreactive to antibody against yolk protein YP2 produced in the follicle cells surrounding developing oocytes. We cloned the fragment of <it>yp2 </it>cDNA from <it>S. littoralis </it>and determined that it is expressed in both ovaries and testes. <it>yp2 </it>mRNA and YP2 protein were detected in the somatic cyst cells enveloping sperm inside the testis. During the period of sperm release, YP2 protein appears in the seminal fluid and forms an external coat on spermatozoa.</p> <p>Conclusion</p> <p>One of the yolk protein precursors YP2, which in females accumulate in the oocytes to provision developing embryos, appears to have a second male-specific role. It is produced in the testes and released into the seminal fluid where it interacts with sperm. These data reveal unexpected common factor in the maturation of insect eggs and sperm.</p

Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and Adjacent Inner Dynein Arms in Tetrahymena Cilia

Motile cilia and eukaryotic flagella are specific cell protrusions that are conserved from protists to humans. They are supported by a skeleton composed of uniquely organized microtubules&mdash;nine peripheral doublets and two central singlets (9 &times; 2 + 2). Microtubules also serve as docking sites for periodically distributed multiprotein ciliary complexes. Radial spokes, the T-shaped ciliary complexes, repeat along the outer doublets as triplets and transduce the regulatory signals from the cilium center to the outer doublet-docked dynein arms. Using the genetic, proteomic, and microscopic approaches, we have shown that lack of Tetrahymena Cfap91 protein affects stable docking/positioning of the radial spoke RS3 and the base of RS2, and adjacent inner dynein arms, possibly due to the ability of Cfap91 to interact with a molecular ruler protein, Ccdc39. The localization studies confirmed that the level of RS3-specific proteins, Cfap61 and Cfap251, as well as RS2-associated Cfap206, are significantly diminished in Tetrahymena CFAP91-KO cells. Cilia of Tetrahymena cells with knocked-out CFAP91 beat in an uncoordinated manner and their beating frequency is dramatically reduced. Consequently, CFAP91-KO cells swam about a hundred times slower than wild-type cells. We concluded that Tetrahymena Cfap91 localizes at the base of radial spokes RS2 and RS3 and likely plays a role in the radial spoke(s) positioning and stability