Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

Gitelman, Stephen E.; Witman, George B.

doi:10.1083/jcb.87.3.764

Cited by 136 publications

(64 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Calmodulin has been identified in the axoneme (8), and, like the 75-kD proteins, it does not appear to be a component of the dynein arms, radial spokes, or the central pair complex, and its precise location is unclear (28). It is thus possible that calmodulin and the 75-kD proteins are located together in a calcium-regulatory complex.…”

Section: Discussionmentioning

confidence: 99%

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Horst¹,

Witman²

1993

The Journal of Cell Biology

Self Cite

View full text Add to dashboard Cite

Abstract.A new mutant strain of Chlamydomonas, ptxl, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wildtype cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models, ptxl-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.T He unicellular biflagellate alga Chlamydomonas has been used extensively in the study of flagellar motility. This is due, in part, to the ease with which mutant strains displaying aberrant motility can be produced and analyzed. As a result, the structures which generate motility are well characterized, yet the processes which regulate motility remain obscure. To study the regulation of flagellar motility, we have generated mutant strains of Chlamydomonas which are defective in their ability to phototax.The two flagella of Chlamydomonas can be distinguished based on their position relative to a specialized region of the chloroplast referred to as the eyespot (13). During phototaxis, the beat pattern of the cis-flagellum (the flagellum closest to the eyespot) and the trans-flagelium are modified, resulting in a change in swimming direction relative to the light. This requires that the cis-and trans-flagella respond differentially to the signal generated by photoreception. Previous studies have provided evidence that calcium is required for phototaxis (3,4,22,25,37). Moreover, the two flagella are differentially responsive to calcium, such that the cis-flagellum is more effective at force generation at low calcium 00 -9 M), and the trans-flagellum more effective at higher calcium (10 -7 M) (17). This differential sensitivity of the cis-and trans-flagella to calcium has been proposed to mediate ...

show abstract

Section: Discussionmentioning

confidence: 99%

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Horst¹,

Witman²

1993

The Journal of Cell Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Calmodulin has been characterised in¯agella and cell bodies (Gitelman and Witman 1980). Furthermore, centrin (caltractin), a calcium-binding contractile protein, has been implicated in de¯agellation (Sanders and Salisbury 1994) and has been cloned (Huang et al 1988).…”

Section: Introductionmentioning

confidence: 99%

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

Siderius

Henskens

Porto-Leblanche

et al. 1997

Planta

View full text Add to dashboard Cite

General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 12 May 2018Abstract. Calcium-stimulated protein kinase activity in the¯agella of the green alga Chlamydomonas moewusii (Gerlo ) was characterised. Using SDS-PAGE and an on-blot phosphorylation assay, a 65-kDa protein was identi®ed as the major calcium-stimulated protein kinase. Its activity was directly stimulated by calcium, a characteristic of the calmodulin-like domain protein kinases (CDPKs). Monoclonal antibodies raised against the CDPKa from soybean cross-reacted with the 65-kDa protein in the¯agella, and also with other proteins in thē agellum and cell body. The same monoclonal antibodies were used to screen a C. moewusii cDNA expression library in order to isolate CDPK cDNAs from C. moewusii. The CCK1 cDNA encodes a protein with a kinase and calmodulin-like domain linked by a junction domain typical of CDPKs. From Southern analyses, evidence was obtained for a CDPK gene family in C. moewusii and C. reinhardtii.

show abstract

“…These include a light chain (LC4) of outer arm dynein (King and Patel-King, 1995); an outer arm docking complex protein (DC3) (Casey et al, 2003); centrin, which is associated with a subset of inner dynein arms (Piperno et al, 1992); calmodulin (Gitelman and Witman, 1980;Van Eldik et al, 1980;Witman and Minervini, 1982; for review, see Otter, 1989); and the protofilament ribbon component Rib72 Ikeda et al, 2003). Identification of Ca 2ϩ -binding proteins associated with individual dynein heavy chains, and analysis of the microtubule-binding properties of mutant dyneins lacking individual motor units (Sakato and King, 2003) suggests that Ca 2ϩ signals may impinge directly on particular heavy chains.…”

Section: Molecular Biology Of the Cell 3898mentioning

confidence: 99%

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase

Patel‐King

Gorbatyuk

Takebe

et al. 2004

MBoC

104

View full text Add to dashboard Cite

The radial spokes are required for Ca 2؉ -initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca 2؉ -independent manner, whereas IQ2 and IQ3 show Ca 2؉ -sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca 2؉ . This Ca 2؉ -responsive enzyme, which accounts for ϳ45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca 2؉ -controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes. INTRODUCTIONThe eukaryotic cilium/flagellum is a highly conserved motile organelle built around nine outer doublet microtubules and a central pair of singlet microtubules that form the 9 ϩ 2 axoneme. Individual flagella provide for the motility of single cells such as mammalian sperm and Chlamydomonas, and ciliated epithelia are essential for fluid/mucus transport in many organs. Moreover, nodal cilia are required to define the left-right axis during mammalian development (Supp et al., 1997;Nonaka et al., 1998). Many cells also contain primary cilia that seem to play critical sensory roles during normal tissue function and development . The power for ciliary/flagellar movement is generated by the inner and outer rows of dynein arms that associate with the A tubule of each microtubule doublet and transiently interact with the B tubule of the adjacent doublet to produce a linear force vector that results in the microtubules sliding with respect to each other. This sliding motion is then converted to a flagellar bend by other axonemal components, including the radial spoke and central pair microtubule complexes (see Mitchell (2000) for a review of Chlamydomonas flagellar structure and function).In Chlamydomonas, control of flagellar waveform is mediated through Ca 2ϩ signaling pathways (Bessen et al., 1980;Kamiya and Witman, 1984). Reactivatio...

show abstract

Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

Cited by 136 publications

References 38 publications

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase

Contact Info

Product

Resources

About

Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

Cited by 136 publications

References 38 publications

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

Contact Info

Product

Resources

About

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase