The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

Abstract

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation. INTRODUCTIONCentrioles are evolutionarily ancient organelles, possibly extant in a common ancestor of all eukaryotic cells (CavalierSmith, 2002). They are required for the organization of interphase and mitotic microtubule arrays and as basal bodies they serve as templates for the growth of nine doublet microtubules in axonemes of cilia and flagella (for review see Marshall, 2007). Although flowering plants and more derived fungi have reduced or eliminated centrioles and cilia, metazoans have diversified their functions in numerous sensory and developmental processes (for reviews see Singla and Reiter, 2006;Dawe et al., 2007). Several mammalian genetic disorders have been linked to defects in centrioles or cilia (for review see Afzelius, 2004;Pan et al., 2005). For example, the proteins associated with Bardet-Biedl syndrome, a debilitating pleiotropic human disorder characterized by obesity, renal abnormalities, polydactyly, retinal degeneration, and genitourinary tract malformations have been shown to localize to or around centrioles and cilia (for review see Beales, 2005;Blacque and Leroux, 2006).Centrioles in actively dividing cells show conservative replication and semiconservative segregation during division (for review see Dawe et al., 2007). As a result, each pair of centrioles consists of an older and a younger centriole that differ in age by at least one cell generation. The "mother" centriole differs both in structure and function from the "daughter" centriole. For example, in the single pair of centrioles found in most types of mammalian cells, the mother centriole nucleates the growth of the primary cilium and contains proteins not present in the daughter centriole. The daughter centriole becomes competent for ciliary assembly only in the ensuing cell cycle (for review see Bornens, 2002). Extension of the pathway for sequential maturation of basal bodies has been ...

Eukaryotic Flagella: Variations in Form, Function, and Composition during Evolution

“…The green alga Chlamydomonas reinhardtii, which since the 1950s has served as the principal model for biologists studying flagellum assembly and function, is a prime example: prior to cell division its flagellar basal bodies uncouple from their associated axonemes and migrate to the nuclear periphery, defining opposite poles of the mitotic spindle (Piasecki et al 2008). Thus, to consider the origin of the flagellum compartment fully one might consider (i) the origin of the centriole; (ii) the origin of nine-fold symmetry; (iii) the evolution of an assembly pathway for flagellum biogenesis; (iv) the initial, and potentially the subsequent, evolutionary advantage(s) offered by a flagellum or flagella.…”

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

“…To gain insights into the 'breast-stroke' motility [1,[9][10][11], mutants in the radial spokes [10], the central pair microtubules [12], dynein arms [8,[13][14][15][16], striated fibers [17], basal bodies [18][19][20], and a battery of these [21] have been useful. Two types of dynein arm mutants have been used to show that the outer dynein arms provide power to the flagellar beat while the inner arm dyneins are essential for normal motility [13,14].…”

TheUNI1andUNI2Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium inChlamydomonas

“…In each cell cycle, the younger basal body, positioned cis to the eyspot, undergoes transformation to become an older basal body, positioned trans to the eyespot (Holmes This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -09 -0900) on November 12, 2008. and Dutcher, 1989). The uni1, uni2, and uni3 mutations preferentially affect the growth of a flagellum from the younger of the two basal bodies (Huang et al, 1982;Dutcher and Trabuco, 1998;Piasecki et al, 2008). These mutations also affect the ability of basal bodies to properly assemble a transition zone (TZ), the region just distal to the point of triplet to doublet microtubule transition (Huang et al, 1982;O'Toole et al, 2003;Piasecki et al, 2008).…”

“…The uni1, uni2, and uni3 mutations preferentially affect the growth of a flagellum from the younger of the two basal bodies (Huang et al, 1982;Dutcher and Trabuco, 1998;Piasecki et al, 2008). These mutations also affect the ability of basal bodies to properly assemble a transition zone (TZ), the region just distal to the point of triplet to doublet microtubule transition (Huang et al, 1982;O'Toole et al, 2003;Piasecki et al, 2008). Mutations in either the UNI1 or the UNI2 gene do not appear to affect triplet microtubule assembly in basal bodies as in the uni3 mutant, but rather result in similarly aberrant and elongated TZ structures (Huang et al, 1982;Piasecki et al, 2008).…”

“…These mutations also affect the ability of basal bodies to properly assemble a transition zone (TZ), the region just distal to the point of triplet to doublet microtubule transition (Huang et al, 1982;O'Toole et al, 2003;Piasecki et al, 2008). Mutations in either the UNI1 or the UNI2 gene do not appear to affect triplet microtubule assembly in basal bodies as in the uni3 mutant, but rather result in similarly aberrant and elongated TZ structures (Huang et al, 1982;Piasecki et al, 2008). The UNI2 gene was shown to encode an alanine-rich phosphoprotein that localizes to both basal bodies and probasal bodies (Piasecki et al, 2008).…”