The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.

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The role of the flagellar transition region: inferences from the analysis of a Chlamydomonas mutant with defective transition region structures

Journal of Cell Science ◽

10.1242/jcs.99.4.731 ◽

1991 ◽

Vol 99 (4) ◽

pp. 731-740

Author(s):

JONATHAN W. JARVIK ◽

JOSEPH P. SUHAN

Keyword(s):

Transition Region ◽

Basal Body ◽

Wild Type ◽

Central Pair ◽

Normal Transition ◽

Thin Section Electron Microscopy ◽

Concomitant Reduction ◽

Section Electron ◽

Type Variable

Thin-section electron microscopy of the Chlamydomonas reinhardtii mutant vfl-2 revealed striking defects in the transition region between basal body and flagellum. In place of the highly organized transition cylinders and stellate fibers characteristic of wild type, variable quantities of poorly organized electron-dense material were present. In many cases the transition region was penetrated by central pair microtubules that passed from the axoneme into the basal body. On the basis of these observations we propose that an important function of the structures present in the normal transition region is to physically exclude the central pair microtubules from the basal body. The transition region is the site of flagellar autotomy – the process by which doublet microtubules are severed and flagella are released from the cell. It has been claimed that autotomy is caused by contraction of the centrin-containing stellate fibers, resulting in the mechanical severing of the doublet microtubules and a concomitant reduction of the diameter of the axoneme adjacent to the abscission point. Our observations do not support this claim in that vfl-2 cells, which lack organized stellate fibers, display effective autotomy unaccompanied by detectable narrowing of the axoneme.

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Evolutionary expansion of poly-aspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00265.2021 ◽

2021 ◽

Author(s):

Anna Orekhova ◽

Balazs Csaba Nemeth ◽

Zsanett Jancso ◽

Andrea Geisz ◽

Dora Mosztbacher ◽

...

Keyword(s):

Double Mutant ◽

Early Age ◽

Hereditary Pancreatitis ◽

Wild Type ◽

Trypsinogen Activation ◽

Histological Damage ◽

Cationic Trypsinogen ◽

Activation Peptide ◽

Aspartate Residue

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen orthologs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased autoactivation of T7 trypsinogen 3-fold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side-chains are preferred for maximal autoactivation and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by N-terminal truncation with chymotrypsin C reduced autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable to that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the poly-aspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.

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A 210 kDa protein is located in a membrane-microtubule linker at the distal end of mature and nascent basal bodies

Journal of Cell Science ◽

10.1242/jcs.112.11.1633 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1633-1644 ◽

Cited By ~ 5

Author(s):

K.F. Lechtreck ◽

A. Teltenkotter ◽

A. Grunow

Keyword(s):

Basal Body ◽

Body Position ◽

Immunogold Electron Microscopy ◽

Basal Bodies ◽

Western Blots ◽

Whole Cells ◽

Extracellular Matrix Components ◽

Green Flagellate ◽

Protein Rich ◽

Novel Protein

A monoclonal antibody raised against purified flagellar basal apparatuses from the green flagellate Spermatozopsis similis reacted with a protein of 210 kDa (p210) in western blots. The protein was partially cloned by immunoscreening of a cDNA library. The sequence encoded a novel protein rich in alanine (25%) and proline (20%), which contained regions similar to proteins of comparable amino acid composition such as extracellular matrix components or the membrane-cytoskeletal linker synapsin. Using a polyclonal antibody (anti-p210) raised against the C-terminal part of p210, it was shown that the protein was highly enriched in the basal apparatuses. Immunogold electron microscopy of isolated cytoskeletons or whole cells revealed that p210 was located in the flagellar transition region. The protein was part of the Y-shaped fibrous linkers between the doublet microtubules and the flagellar membrane, as indicated by statistical analysis of post-labeled sections using anti-centrin and anti-tubulin as controls. In premitotic cells p210 was located in a fibrous layer at the distal end of nascent basal bodies, which was perforated by the outgrowing axoneme. During deflagellation the protein remained at the basal body but we observed changes in its distribution, indicating that p210 partially moved to the tip of the basal body. p210 can be used as a marker to determine basal body position, orientation (parallel or antiparallel) and number in S. similis by indirect immunofluorescence. We suppose that p210 is involved in linking basal bodies to the plasma membrane, which is an important step during ciliogenesis.

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Development of surface pattern during division in Paramecium. I. Mapping of duplication and reorganization of cortical cytoskeletal structures in the wild type

Development ◽

10.1242/dev.105.2.191 ◽

1989 ◽

Vol 105 (2) ◽

pp. 191-211 ◽

Cited By ~ 1

Author(s):

F. Iftode ◽

J. Cohen ◽

F. Ruiz ◽

A.T. Rueda ◽

L. Chen-Shan ◽

...

Keyword(s):

Basal Body ◽

Surface Pattern ◽

Basal Bodies ◽

Wild Type ◽

Cell Level ◽

Local Constraints ◽

Whole Cell ◽

Cortical Unit ◽

Asymmetrical Pattern ◽

In The Wild

The shape of a Paramecium is determined by the organization of its cortex which constitutes most of the cell cytoskeleton. These structures and networks are organized in relation to the approx. 4000 ciliary basal bodies present at the surface. Each basal body is the centre of a polarized and asymmetrical cortical unit. At the whole-cell level, all units are tandemly arranged in parallel rows and form a defined asymmetrical pattern with dorsoventral and anteroposterior polarities. During division, the cortex is the site of the major morphogenetic processes. In order to analyse how the surface pattern and the shape of the cell are reconstructed at each division, we have used specific immunological and cytological probes to map, in space and time, the reorganization of each of the major cytoskeletal cortical components: basal bodies and microtubules, kinetodesmal fibres, epiplasm and outer lattice. This cytological dissection demonstrates that the surface of the dividing cell is progressively invaded by morphogenetic waves which successively and individually trigger the duplication, assembly or reorganization of each structure and which all spread from the same epicentre (oral apparatus and fission furrow) with the same shape. Furthermore, the response of units to the morphogenetic waves depends on their position on the cell. It thus appears that despite the structural local constraints within units, the development of surface pattern is controlled in an integrated manner by transcellular signals.

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Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6805 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6805-6815 ◽

Cited By ~ 88

Author(s):

Jens Solsbacher ◽

Patrick Maurer ◽

F. Ralf Bischoff ◽

Gabriel Schlenstedt

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Nuclear Pore ◽

Wild Type ◽

Importin Α ◽

Localization Signal ◽

Green Fluorescent ◽

Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

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Enhancement of Fluoroquinolone Activity by C-8 Halogen and Methoxy Moieties: Action against a Gyrase Resistance Mutant of Mycobacterium smegmatis and a Gyrase-Topoisomerase IV Double Mutant of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2703-2709.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2703-2709 ◽

Cited By ~ 34

Author(s):

Tao Lu ◽

Xilin Zhao ◽

Xinying Li ◽

Alex Drlica-Wagner ◽

Jian-Ying Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mycobacterium Smegmatis ◽

Double Mutant ◽

Structure Refinement ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Topoisomerase Iv ◽

Methoxy Groups ◽

Bactericidal Activities ◽

Mutant Cells

ABSTRACT The increasing prevalence of antibiotic resistance among bacterial pathogens prompted a microbiological study of fluoroquinolone structure-activity relationships with resistant mutants. Bacteriostatic and bactericidal activities for 12 fluoroquinolones were examined with a gyrase mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus. For both organisms C-8 halogen and C-8 methoxy groups enhanced activity. The MIC at which 99% of the isolates tested were inhibited (MIC99) was reduced three- to fivefold for the M. smegmatis mutant and seven- to eightfold for theS. aureus mutant by C-8 bromine, chlorine, and methoxy groups. With both organisms a smaller reduction in the MIC99 (two- to threefold) was associated with a C-8 fluorine moiety. In most comparisons with M. smegmatis the response to a C-8 substituent was similar (within twofold) for wild-type and mutant cells. In contrast, mutant S. aureuswas affected more than the wild type by the addition of a C-8 substituent. C-8 halogen and methoxy groups also improved the ability to kill the two mutants and the respective wild-type cells when measured with various fluoroquinolone concentrations during an incubation period equivalent to four to five doubling times. Collectively these data help define a group of fluoroquinolones that can serve (i) as a base for structure refinement and (ii) as test compounds for slowing the development of fluoroquinolone resistance during infection of vertebrate hosts.

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The role of palmitoylation of the guanine nucleotide binding protein G11α in defining interaction with the plasma membrane

Biochemical Journal ◽

10.1042/bj3101021 ◽

1995 ◽

Vol 310 (3) ◽

pp. 1021-1027 ◽

Cited By ~ 32

Author(s):

J F McCallum ◽

A Wise ◽

M A Grassie ◽

A I Magee ◽

F Guzzi ◽

...

Keyword(s):

Double Mutant ◽

Sodium Cholate ◽

Particulate Fraction ◽

Wild Type ◽

Guanine Nucleotide Binding Protein ◽

Sds Page ◽

Exclusion Chromatography ◽

Resistant Mutants ◽

Guanine Nucleotide Binding

Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)

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The UDP-Glc:Glycoprotein Glucosyltransferase Is Essential for Schizosaccharomyces pombe Viability under Conditions of Extreme Endoplasmic Reticulum Stress

The Journal of Cell Biology ◽

10.1083/jcb.143.3.625 ◽

1998 ◽

Vol 143 (3) ◽

pp. 625-635 ◽

Cited By ~ 57

Author(s):

Sandra Fanchiotti ◽

Fabiana Fernández ◽

Cecilia D'Alessio ◽

Armando J. Parodi

Keyword(s):

Cell Viability ◽

Schizosaccharomyces Pombe ◽

Double Mutant ◽

Expression Vector ◽

The Other ◽

Wild Type ◽

Glucosidase Ii ◽

Wild Type Phenotype ◽

Polypeptide Chains ◽

Mutant Cells

Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28°C. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28°C and did not grow at 37°C. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37°C and had, when grown at 28°C, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation–deglucosylation catalyzed by GT and GII.

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Fatty Acid Remodeling of GPI-anchored Proteins Is Required for Their Raft Association

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0885 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1497-1506 ◽

Cited By ~ 144

Author(s):

Yusuke Maeda ◽

Yuko Tashima ◽

Toshiaki Houjou ◽

Morihisa Fujita ◽

Takehiko Yoko-o ◽

...

Keyword(s):

Fatty Acid ◽

Lipid Rafts ◽

Cho Cells ◽

Membrane Fraction ◽

Double Mutant ◽

Chinese Hamster ◽

Wild Type ◽

Saturated Chain ◽

Mutant Cells ◽

Detergent Resistant Membrane

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3. The surface GPI-APs isolated from the PGAP2 and -3 double-mutant Chinese hamster ovary (CHO) cells had unsaturated chains, such as oleic, arachidonic, and docosatetraenoic acids in the sn-2 position, whereas those from wild-type CHO cells had exclusively stearic acid, a saturated chain, indicating that the sn-2 chain is exchanged to a saturated chain. We then assessed the association of GPI-APs with lipid rafts. Recovery of unremodeled GPI-APs from the double-mutant cells in the detergent-resistant membrane fraction was very low, indicating that GPI-APs become competent to be incorporated into lipid rafts by PGAP3- and PGAP2-mediated fatty acid remodeling. We also show that the remodeling requires the preceding PGAP1-mediated deacylation from inositol of GPI-APs in the endoplasmic reticulum.

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Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.05153-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3273-3283 ◽

Cited By ~ 58

Author(s):

Syed Z. Sultan ◽

Joshua E. Pitzer ◽

Tristan Boquoi ◽

Gerry Hobbs ◽

Michael R. Miller ◽

...

Keyword(s):

Life Cycle ◽

Borrelia Burgdorferi ◽

Double Mutant ◽

Ixodes Scapularis ◽

Metabolizing Enzymes ◽

Content Type ◽

Motility Regulation ◽

Mutant Cells ◽

Single Set

ABSTRACTHD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria.Borrelia burgdorferipossesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation inpdeAresulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with aKmof 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing thepdeA pdeBdouble mutant, we demonstrate that no additional phosphodiesterases are present inB. burgdorferi.pdeBsingle mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing apilZpdeBdouble mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly inpdeBmutant cells, these cells exhibited a reduced ability to survive inIxodes scapularisticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role ofpdeBincreases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle ofB. burgdorferi.

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