scholarly journals Functional evidence for in vitro microtubule severing by the plant katanin homologue

2002 ◽  
Vol 365 (2) ◽  
pp. 337-342 ◽  
Author(s):  
Virginie STOPPIN-MELLET ◽  
Jérémie GAILLARD ◽  
Marylin VANTARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Plant Cells ◽  
Microtubule Assembly ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Microtubule Severing ◽  
Thale Cress ◽  
Temporal And Spatial

Temporal and spatial assembly of microtubules in plant cells depends mainly on the activity of microtubule-interacting proteins, which either stabilize, destabilize or translocate microtubules. Recent data have revealed that the thale cress (Arabidopsis thaliana) contains a protein related to the p60 catalytic subunit of animal katanin, a microtubule-severing protein. However, effects of the plant p60 on microtubule assembly are not known. We report the first functional evidence that the recombinant A. thaliana p60 katanin subunit, Atp60, binds to microtubules and severs them in an ATP-dependent manner in vitro. ATPase activity of Atp60 is stimulated by low tubulin/katanin ratios, and is inhibited at higher ratios. Considering its properties in vitro, several functions of Atp60 in vivo are discussed.

Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis

2004 ◽  
Vol 287 (4) ◽  
pp. F602-F611 ◽  
Author(s):  
Dong Chen ◽  
Richard Roberts ◽  
Martin Pohl ◽  
Sanjay Nigam ◽  
Jordan Kreidberg ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Branching Morphogenesis ◽  
Integrin Subunit ◽  
Integrin Expression ◽  
Dependent Manner ◽  
Ureteric Bud ◽  
Laminin Receptors ◽  
Temporal And Spatial

Inner medullary collecting ducts (IMCD) are terminally differentiated structures derived from the ureteric bud (UB). UB development is mediated by changes in the temporal and spatial expression of integrins and their respective ligands. We demonstrate both in vivo and in vitro that the UB expresses predominantly laminin receptors (α3β1-, α6β1-, and α6β4-integrins), whereas the IMCD expresses both collagen (α1β1- and α2β1-integrins) and laminin receptors. Cells derived from the IMCD, but not the UB, undergo tubulogenesis in collagen-I (CI) gels in an α1β1- and α2β1-dependent manner. UB cells transfected with the α2-integrin subunit undergo tubulogenesis in CI, suggesting that collagen receptors are required for branching morphogenesis in CI. In contrast, both UB and IMCD cells undergo tubulogenesis in CI/Matrigel gels. UB cells primarily utilize α3β1- and α6-integrins, whereas IMCD cells mainly employ α1β1 for this process. These results demonstrate a switch in integrin expression from primarily laminin receptors in the early UB to both collagen and laminin receptors in the mature IMCD, which has functional consequences for branching morphogenesis in three-dimensional cell culture models. This suggests that temporal and spatial changes in integrin expression could help organize the pattern of branching morphogenesis of the developing collecting system in vivo.

Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression

2011 ◽  
Vol 436 (3) ◽  
pp. 547-557 ◽  
Author(s):  
Xuebin Zhang ◽  
Carine De Marcos Lousa ◽  
Nellie Schutte-Lensink ◽  
Rob Ofman ◽  
Ronald J. Wanders ◽  
...  
Keyword(s):  
Quality Control ◽  
Arabidopsis Thaliana ◽  
Abc Transporters ◽  
Plant Cells ◽  
Related Protein ◽  
Peroxisomal Membrane ◽  
And Function ◽  
Native Host

ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some ‘protein-absent’ mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid β-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.

scholarly journals Synapsins as major neuronal Ca2+/S100A1-interacting proteins

1999 ◽  
Vol 344 (2) ◽  
pp. 577-583 ◽  
Author(s):  
Jörg HEIERHORST ◽  
Ken I. MITCHELHILL ◽  
Richard J. MANN ◽  
Tony TIGANIS ◽  
Andrew J. CZERNIK ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Double Labelling ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Brain Extracts ◽  
Dependent Protein

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

scholarly journals Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction

2008 ◽  
Vol 416 (2) ◽  
pp. 289-296 ◽  
Author(s):  
Adrian Alder ◽  
Iris Holdermann ◽  
Peter Beyer ◽  
Salim Al-Babili
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Oryza Sativa ◽  
Plant Hormone ◽  
Dependent Manner ◽  
Cleavage Reaction ◽  
Cleavage Step ◽  
High Tillering

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds β-apo-10′-carotenal and its alcohol into β-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.

scholarly journals Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

2008 ◽  
Vol 413 (3) ◽  
pp. 447-457 ◽  
Author(s):  
Jakob Wiborg ◽  
Charlotte O'Shea ◽  
Karen Skriver
Keyword(s):  
Arabidopsis Thaliana ◽  
Large Family ◽  
Degradation Pathway ◽  
Prolyl Isomerase ◽  
Thale Cress ◽  
Binding Cleft ◽  
Ubiquitin Conjugating Enzymes ◽  
Protein Ligases

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp266 in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis–trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis–trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3–E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

scholarly journals Blocking cell microtubule assembly inhibits the alloimmune response in vitro and prolongs renal allograft survival by inhibition of Th1 and sparing of Th2 cell function in vivo.

1995 ◽  
Vol 5 (7) ◽  
pp. 1418-1425
Author(s):  
E Akalin ◽  
W W Hancock ◽  
N Perico ◽  
G Remuzzi ◽  
O Imberti ◽  
...  
Keyword(s):  
Microtubule Assembly ◽  
Brown Norway ◽  
Cytotoxic T Cell ◽  
Dependent Manner ◽  
Lymphocyte Response ◽  
Term Survival ◽  
Renal Allografts ◽  
Dose Dependent

Colchicine inhibits cell microtubule assembly by binding to and preventing the polymerization of tubulin monomers. Although there are data to indicate that colchicine inhibits a variety of cell-mediated immune responses, the effects and mechanisms of inhibiting cell microtubule assembly on the alloimmune response have not been thoroughly investigated. It has recently been shown that colchicine prevents acute rejection and promotes the long-term survival of rat renal allografts. In this study, the effects and mechanisms of inhibiting cell microtubule assembly by colchicine on the alloimmune response in vitro and in vivo were examined. First, the effects of colchicine on T lymphocyte response to alloantigen in vitro were tested. In the standard one-way mixed lymphocyte response (MLR), responder Lewis rat lymph node cells were cultured with irradiated Brown-Norway stimulators. Colchicine inhibited the MLR in a dose-dependent manner, with 100% inhibition at a concentration of 25 ng/mL (6.25 x 10(-8) M) and 50% inhibition at a concentration of approximately 5 to 10 ng/mL. Colchicine also inhibited the generation of cytotoxic T lymphocytes as well as cytotoxic T cell effector function in vitro in a dose-dependent fashion. Second, detailed immunohistologic studies of renal allografts harvested from unmodified control (acutely rejecting) and colchicine-treated rats (Day 15 or 30) were performed. These studies showed that grafts from colchicine-treated animals had significantly fewer mononuclear cell infiltrates and less edema, and moderately decreased deposition of immunoglobulin M, C3, and fibrin, as compared with acutely rejecting control grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Microtubule bundling by MAP65-1 protects against severing by inhibiting the binding of katanin

2019 ◽  
Author(s):  
Graham M Burkart ◽  
Ram Dixit
Keyword(s):  
Cortical Microtubule ◽  
Structural Features ◽  
Dependent Manner ◽  
Protective Mechanisms ◽  
Concentration Dependent Manner ◽  
Mutant Proteins ◽  
Microtubule Severing ◽  
Biological Studies

ABSTRACTThe microtubule-severing enzyme katanin regulates the organization and turnover of microtubule arrays by the localized breakdown of microtubule polymers. In land plants, katanin (KTN1) activity is essential for the formation of linearly organized cortical microtubule arrays which determine the axis of cell expansion. Cell biological studies have shown that even though KTN1 binds to the sidewalls of single and bundled microtubules, severing activity is restricted to microtubule crossover and nucleation sites, indicating that cells contain protective mechanisms to prevent indiscriminate microtubule severing. Here, we show that the microtubule bundling protein MAP65-1 inhibits KTN1-mediated microtubule severing in vitro. Severing is inhibited at bundled microtubule segments and the severing rate of non-bundled microtubules is reduced by MAP65-1 in a concentration-dependent manner. Using various MAP65-1 mutant proteins, we demonstrate that efficient crosslinking of microtubules is crucial for this protective effect and that microtubule binding alone is not sufficient. Reduced severing due to microtubule bundling by MAP65-1 correlated to decreased binding of KTN1 to these microtubules. Taken together, our work reveals that crosslinking of microtubules by MAP65-1 confers resistance to severing by inhibiting the binding of katanin and identifies the structural features of MAP65-1 that are important for this activity.Highlight SummaryCortical microtubule bundles resist severing in vivo. Here, we show that crosslinking of microtubules by MAP65-1 inhibits severing in a dose-dependent manner by preventing katanin from binding to these microtubules.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

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