Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo

Cytoskeleton ◽  
2010 ◽  
Vol 67 (7) ◽  
pp. 466-476 ◽  
Author(s):  
Hiroko Takazaki ◽  
Zhongmei Liu ◽  
Mingyue Jin ◽  
Ritsu Kamiya ◽  
Takuo Yasunaga
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Heavy Chains ◽  
Dynein Heavy Chains

scholarly journals Identification of parallel pH- and zeaxanthin-dependent quenching of excess energy in LHCSR3 in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Julianne M. Troiano ◽  
Federico Perozeni ◽  
Raymundo Moya ◽  
Luca Zuliani ◽  
Kwangryul Baek ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Light Harvesting ◽  
Complex Stress ◽  
Excess Energy ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Light Harvesting Complex ◽  
Non Photochemical Quenching

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

In Vivo and in Vitro Studies on the Interaction Between Cytochrome f and Plastocyanin in Chlamydomonas reinhardtii

1998 ◽  
pp. 1589-1592 ◽  
Author(s):  
Luis R. Comolli ◽  
Jianhui Zhou ◽  
Thomas Linden ◽  
Rainer Breitling ◽  
Jorge Flores ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
In Vitro Studies ◽  
Cytochrome F

scholarly journals In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

1995 ◽  
Vol 6 (3) ◽  
pp. 283-296 ◽  
Author(s):  
L M Hendershot ◽  
J Y Wei ◽  
J R Gaut ◽  
B Lawson ◽  
P J Freiden ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Binding ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Atp Binding ◽  
Heavy Chains ◽  
In Vivo Expression

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

scholarly journals Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella

2009 ◽  
Vol 186 (3) ◽  
pp. 437-446 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Heavy Chain ◽  
Single Particle ◽  
Heavy Chains ◽  
Molecular Arrangement ◽  
Dynein Arms ◽  
Electron Cryotomography ◽  
Microtubule Doublet ◽  
Asymmetrical Bending ◽  
Dynein Heavy Chains

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

A New Class of Hexavalent Bispecific Antibodies and Immunocytokines with Enhanced Pharmacokinetics and Improved Efficacy in Vivo.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2451-2451
Author(s):  
Edmund A Rossi ◽  
Thomas M Cardillo ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
Light Chain ◽  
Fusion Proteins ◽  
Low Dose ◽  
Molecular Size ◽  
Bispecific Antibodies ◽  
Heavy Chains ◽  
New Class ◽  
Anti Cd20

Abstract Abstract 2451 Background. Biomedical research is trending to the development of increasingly more sophisticated antibody-based biologics, such as bispecific antibodies, immunocytokines and antibody-drug conjugates. Compared to traditional mAbs, development of more complex, and less natural, fusion proteins are challenged by problems with yield, stability, toxicity, immunogenicity and Pk. Previously, we reported potent anti-lymphoma activity for both anti-CD22/CD20 bispecific hexavalent antibodies (bsHexAbs; Rossi et al., Blood 2009; 113: 6161–6171) and immunocytokines comprising anti-CD20 mAb and tetrameric IFNα2b (IgG-IFN; Rossi et al., Blood 2009;114:3864-71). For each class of immunoconjugate that were produced with the Dock-and-Lock (DNL) method using an IgG module having an AD2 peptide fused at the C-terminal end of the Fc, we identified Pk and in vivo stability as potentially limiting parameters. Methods. Using the DNL method, we generated a new class of IgG modules, which have an AD2 peptide fused at the C-terminal end of the kappa light chain and were used to produce Ck-based (indicated by *) bsHexAbs and IgG-IFNα2b, for comparison with homologous Fc-based constructs. The Ck-based immunocytokine 20*-2b, has a similar molecular size and composition to its Fc-based homolog, 20-2b, each comprising the humanized anti-CD20 mAb, veltuzumab, and 4 IFNα2b groups that are fused at the C-terminal ends of the light or heavy chains, respectively. The Ck-based bsHexAb 22*-(20)-(20) and its Fc-based homolog 22-(20)-(20), each comprise the humanized anti-CD22 mAb, epratuzumab, and 4 Fabs of veltuzumab, which are fused at the C-terminal ends of the light and heavy chains, respectively. Results. The Ck-based constructs exhibited superior Pk (longer T1/2) in mice and rabbits, with either IV or SC injection (Table 1). Although the bsHexAbs and IgG-IFN are considerably stable in sera, analysis of Pk samples indicated that some dissociation occurs in vivo, presumably by intracellular processing. The in vivo dissociation rate for 20*-2b (0.18%/h) was 5.4-fold slower than 20-2b (0.97%/h). Similarly, 22*-(20)-(20) (0.19%/h) was more stable in vivo than 22-(20)-(20) (0.55%/h). Fc effector functions were markedly enhanced for the Ck-based constructs. Where 20*-2b induced strong CDC, which approached the potency of veltuzumab, no activity was evident for 20-2b. Epratuzumab did not have CDC, while 22-(20)-(20) achieved modestly increased activity, and 22*-(20)-(20) induced even greater CDC. In vitro, epratuzumab induced minimal ADCC and 22-(20)-(20) did not show a statistically significant improvement. However, 22*-(20)-(20) exhibited potent ADCC, which was similar to that of veltuzumab. Finally, the Ck-based conjugates were more effective than the Fc-based counterparts for therapy of disseminated NHL (Daudi) xenografts. Using a single low dose of 0.25 mg, superiority (P=0.0351) was demonstrated for 20*-2b (median survival time [MST]>189 days, 87% cures), compared to 20-2b (MST=134.5 days, 37.5% cures). At a high (1 mg) dose, 22*-(20)-(20) (MST>98 days, 100% survival) was superior (P<0.0001) to 22-(20)-(20) (MST=71 days, 10% survival). With low-dose (10 μg) treatment, the MST was 91 days for 22*-(20)-(20), compared to 50.5 days for 22-(20-(20) (P=0.0014). Conclusions. These new constructs demonstrate the further enhancement of two different classes of fusion proteins with already potent anti-lymphoma efficacy. Due to extended Pk, improved stability and enhanced effector function, the Ck-based design is superior for in vivo applications. The strategy of designing antibody fusion proteins at the C-terminus of the light chain, instead of at the commonly used Fc, may improve the in vivo efficacy of most immunoconjugates in general, and the DNL conjugates in particular. Disclosures: Rossi: Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership. Chang:Immunomedics, Inc.: Employment.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii.

1984 ◽  
Vol 98 (1) ◽  
pp. 1-7 ◽  
Author(s):  
F A Wollman ◽  
P Delepelaire
Keyword(s):  
Photosystem Ii ◽  
Chlamydomonas Reinhardtii ◽  
Redox State ◽  
Light Harvesting ◽  
Anaerobic Treatment ◽  
Intact Cells ◽  
Light Harvesting Complex ◽  
Plastoquinone Pool

We have used a new method to extensively modify the redox state of the plastoquinone pool in Chlamydomonas reinhardtii intact cells. This was achieved by an anaerobic treatment that inhibits the chlororespiratory pathway recently described by P. Bennoun (Proc. Natl. Acad. Sci. USA, 1982, 79:4352-4356). A state I (plus 3,4-dichlorophenyl-1,1-dimethylurea) leads to anaerobic state transition induced a decrease in the maximal fluorescence yield at room temperature and in the FPSII/FPSI ratio at 77 degrees K, which was three times larger than in a classical state I leads to state II transition. The fluorescence changes observed in vivo were similar in amplitude to those observed in vitro upon transfer to the light of dark-adapted, broken chloroplasts incubated in the presence of ATP. We then compared the phosphorylation pattern of thylakoid polypeptides in C. reinhardtii in vitro and in vivo using gamma-[32P]ATP and [32P]orthophosphate labeling, respectively. The same set of polypeptides, mainly light-harvesting complex polypeptides, was phosphorylated in both cases. We observed that this phosphorylation process is reversible and is mediated by the redox state of the plastoquinone pool in vivo as well as in vitro. Similar changes of even larger amplitude were observed with the F34 mutant intact cells lacking in photosystem II centers. The presence of the photosystem II centers is then not required for the occurrence of the plastoquinone-mediated phosphorylation of light-harvesting complex polypeptides.

Glycosaminoglycan-bound and free inter-α-trypsin inhibitor components of follicular fluid

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 283-288 ◽  
Author(s):  
Lars Ødum ◽  
Torben E. Jessen ◽  
Claus Yding Andersen
Keyword(s):  
Extracellular Matrix ◽  
Trypsin Inhibitor ◽  
Follicular Fluid ◽  
Normal Female ◽  
Heavy Chains ◽  
Rate Limiting Step ◽  
Fluid Concentration ◽  
Rate Limiting

The proteinase inhibitor inter-α trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.

Sign in / Sign up

Export Citation Format

Share Document