Sas4 links basal bodies to cell division via Hippo signaling

Basal bodies (BBs) are macromolecular complexes required for the formation and cortical positioning of cilia. Both BB assembly and DNA replication are tightly coordinated with the cell cycle to ensure their accurate segregation and propagation to daughter cells, but the mechanisms ensuring coordination are unclear. The Tetrahymena Sas4/CPAP protein is enriched at assembling BBs, localizing to the core BB structure and to the base of BB-appendage microtubules and striated fiber. Sas4 is necessary for BB assembly and cortical microtubule organization, and Sas4 loss disrupts cell division furrow positioning and DNA segregation. The Hippo signaling pathway is known to regulate cell division furrow position, and Hippo molecules localize to BBs and BB-appendages. We find that Sas4 loss disrupts localization of the Hippo activator, Mob1, suggesting that Sas4 mediates Hippo activity by promoting scaffolds for Mob1 localization to the cell cortex. Thus, Sas4 links BBs with an ancient signaling pathway known to promote the accurate and symmetric segregation of the genome.

DisAp-dependent striated fiber elongation is required to organize ciliary arrays

Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp’s similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains of Chlamydomonas reinhardtii

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the δ-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in α-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.

Abnormal basal-body number, location, and orientation in a striated fiber-defective mutant of Chlamydomonas reinhardtii.

We describe a mutant of Chlamydomonas reinhardtii in which basal body associated striated fibers are absent or incomplete. Basal body spacing, angle, and relative rotational orientation are abnormal and extremely variable. Abnormal partitioning of cellular contents at cytokinesis is also evident. Mating, maintenance of flagellar length equality, and backward swimming response are normal. Genetic analysis indicates mutation of a new Mendelian gene--vfl-3--linked to the centromere of Chromosome VI.

“Dirty” Fiber - “Clean” Fiber: A Theory of the Organization of Fibrin from Fibrinogen

SummaryA theory of fibrin formation is proposed in which gel fiber fabrication is coordinated by two distinct sets of complexing filaments emanating from spherical fibrinogen-derived structural subunits. The formulation is an attempt to correlate the previously reported ultramicroscopic negative-contrast images of fibrinogen and fibrin with a similar but unique spheroidal body observed during the organization of dysfibrin Seattle. This slow clotting dysfibrinogen manifested a predisposition to form aperiodic fibers when thrombic proteolysis and polymerization were curtailed by the progressively earlier application of an acid stain embedment reagent (uranyl acetate). The unstriated fibers were unevenly surrounded by the electron dense stain and manifested a “dirty” appearance. This “primordial” fiber was comprised of linear chains of (10 nm) complexed spheres with (15 nm) lateral filamentous projections. Fibrinogen Seattle consistently generated typically striated (26 nm), “clean”, or “mature” fiber gels when polymerization was allowed to proceed. The proposed role of these lateral filaments is to engage, align, and stabilize similar processes on globular subunits in adjacent linear chains of the “primordial” fiber. The behavior of the negative contrast reagent during the fibrinogen to fibrin conversion is assessed as a reflection of the distribution and orientation of polar residues. The“clean” or striated fiber is depicted as a highly fenestrated hollow cylinder with a hydrophilic interior. The model is fitted to ultrastructural studies on the progression of transamidation of fibrin by factor XIIIa.

Interrelationships between microtubules, a striated fiber, and the gametic mating structure of Chlamydomonas reinhardi.

The microtubule system associated with the Chlamydomonas reinhardi flagellar apparatus is shown to differ from previous descriptions; two of the four flagellar "roots" possess only two microtubules and are associated with a finely striated fiber. In gametic cells this fiber underlies the gametic mating structure and makes contact with it. Functional interpretations are offered.