Autotomy of algal flagella: electron microscope studies of Chlamydomonas (Chlorophyceae) and Tetraselmis (Prasinophyceae)

2004

MBoC

Polycystic kidney disease and related syndromes involve dysregulation of cell proliferation in conjunction with ciliary defects. The relationship between cilia and cell cycle is enigmatic, but it may involve regulation by the NIMA-family of kinases (Neks). We previously showed that the Nek Fa2p is important for ciliary function and cell cycle in Chlamydomonas. We now show that Fa2p localizes to an important regulatory site at the proximal end of cilia in both Chlamydomonas and a mouse kidney cell line. Fa2p also is associated with the proximal end of centrioles. Its localization is dynamic during the cell cycle, following a similar pattern in both cell types. The cell cycle function of Fa2p is kinase independent, whereas its ciliary function is kinase dependent. Mice with mutations in Nek1 or Nek8 have cystic kidneys; therefore, our discovery that a member of this phylogenetic group of Nek proteins is localized to the same sites in Chlamydomonas and kidney epithelial cells suggests that Neks play conserved roles in the coordination of cilia and cell cycle progression. INTRODUCTIONThe Nek family of kinases is defined by sequence similarity to NIMA, the Aspergillus kinase that is essential for entry into mitosis (reviewed by Morris and Enos, 1992;O'Connell et al., 2003). Fungi and higher plants encode only one or a few members of this family (Kambouris et al., 1993;Pu et al., 1995;Krien et al., 1998;Wang et al., 2003), but in organisms with centrioles and cilia, such as humans and Chlamydomonas, the family is expanded to 10 or more members (O'Connell et al., 2003;Bradley et al., 2004; Bradley and Quarmby, unpublished data), some of which affect ciliary length (Wloga, Rogowski, and Gaertig, ASCB meeting, 2003, Abstract 2439). We recently reported that the Chlamydomonas Nek Fa2p participates in ciliary function and cell cycle progression (Mahjoub et al., 2002).An emerging pattern suggests that a variety of human syndromes are related to defects in the assembly, maintenance, or function of cilia (Ong and Wheatley, 2003;Pazour and Witman, 2003;Li et al., 2004;Sun et al., 2004). Ciliopathies, including polycystic kidney disease, Bardet-Beidl syndrome, and nephronophthisis include kidney cyst formation as an important component of a pleiotropic syndrome (Mykytyn and Sheffield, 2004;Wilson, 2004). This important consequence of these diseases arises, in part, from dysregulation of cell proliferation in conjunction with ciliary dysfunction. Of particular relevance to the current study are reports that mutations in vertebrate Nek1 and Nek8 cause cystic kidneys in mice and zebrafish (Upadhya et al., 2000;Liu et al., 2002).The relationship between cilia and cell cycle progression is poorly understood. In many cells, entry into the cell cycle is preceded by ciliary disassembly, and exit from mitosis is accompanied by ciliary assembly, a relationship that may reflect the use of the basal bodies/centrioles as mitotic spindle poles (Tucker and Pardee, 1979;Ehler et al., 1995;Wheatley et al., 1996). Consistent with this idea, si...

Section: Discussionmentioning

confidence: 91%

A NIMA-related Kinase, Fa2p, Localizes to a Novel Site in the Proximal Cilia ofChlamydomonasand Mouse Kidney Cells

Mahjoub

Rasi

2004

MBoC

“…This cellular behavior, known as deflagellation or flagellar autotomy, is a highly specific process that involves the severing of the nine outer doublet axonemal microtubules. Electron microscopy reveals that the microtubules are severed at a precise site distal to the transition zone between the axoneme and the basal bodies (Lewin and Lee, 1985;Salisbury, 1989, 1994;Jarvik and Suhan, 1991;Taillon et al, 1992). Previously, we have shown that cytosolic acidification triggers deflagellation via the activation of a Ca 2ϩ influx pathway (Hartzell et al, 1993;Quarmby and Hartzell, 1994;Quarmby, 1996).…”

Section: Introductionmentioning

confidence: 99%

A Role for Katanin-mediated Axonemal Severing duringChlamydomonasDeflagellation

Lohret

McNally

1998

MBoC

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca2+, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of theChlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate thatChlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca2+-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.

“…108:1751-1760. We conclude that mastoparan induces the release of an intracellular pool of Ca 2+ whereas acid induces an influx of extraceUular Ca 2+ to activate the machinery of deflagellation.D FLAGELLATION is a specific event whereby the flagella are precisely excised from the cell body (Rosenbanm and Carlson, 1969;Satir et al, 1976;Lewin and Lee, 1985;Sanders and Salisbury, 1989; Jarvik and Suhan, 1991). The physical mechanism of flagellar excision appears to involve both a microtubule severing activity (Vale, 1991;Shiina et al, 1992;McNally and Vale, 1993) and a mechanical force generated by centrin (for references see Hartzell et al, 1993).…”

mentioning

confidence: 99%

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The Journal of Cell Biology

Hartzell

1994

108

Abstract. The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca 2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca 2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2÷; (b) sensitivity to Ca 2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760. We conclude that mastoparan induces the release of an intracellular pool of Ca 2+ whereas acid induces an influx of extraceUular Ca 2+ to activate the machinery of deflagellation.D FLAGELLATION is a specific event whereby the flagella are precisely excised from the cell body (Rosenbanm and Carlson, 1969;Satir et al., 1976;Lewin and Lee, 1985;Sanders and Salisbury, 1989; Jarvik and Suhan, 1991). The physical mechanism of flagellar excision appears to involve both a microtubule severing activity (Vale, 1991;Shiina et al., 1992;McNally and Vale, 1993) and a mechanical force generated by centrin (for references see Hartzell et al., 1993). A stellate array of centrin-containing transition zone fibers contract during deflagellation (Sanders and Salisbury, 1989). Chlamydomonas cells permeabilized with the non-ionic detergent, NP-40, deflagellate when Ca 2+ is added in /~M concentrations (Sanders and Salisbury, 1989). Because Ca 2÷ is necessary and sufficient for deflagellation in detergent-permeabilized cells (Sanders and Salisbury, 1994), agents which induce deflagellation in vivo may act via increases in intracellular [Ca2+]. Deflagellation can be produced in living cells by a variety of stimuli (Minz and Lewin, 1954;Thompson et al., 1974;Lewin et al., 1980;Witman, 1986). We have previously shown that acid flux into the cell triggers deflagellation in vivo (Hartzell et al., 1993) as does external application of the wasp venom peptide, mastoparan (Quarmby et al., 1992). We now pose the question: How do these agents generate an intracellular Ca 2÷ signal in vivo?Address all correspondence to L, M, Quarmby, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.In the only published report to examine the requirement for extracellular Ca 2+ during acid-induced deflagellation, the authors state that a 30-min pretreatment in [Ca 2+] below 0.1 #M inhibited acid-induced deflagellation, but t...