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21.
Centrin homologues in higher plants are prominently associated with the developing cell plate 总被引:3,自引:0,他引:3
A. J. Del Vecchio J. D. I. Harper K. C. Vaughn A. T. Baron J. L. Salisbury R. L. Overall 《Protoplasma》1997,196(3-4):224-234
Summary Centrin and calmodulin are members of the EF-hand calcium-binding superfamily of proteins. In this study we compared localisation and immunoblotting of centrin with calmodulin in several monocot (onion and wheat) and dicot (mung bean andArabidopsis) plants. We confirmed that an anti-calmodulin antibody recognised a 17 kDa protein in all species tested and localises to the cytoplasm, mitotic matrix and with microtubules of the preprophase band and phragmoplast. In contrast, immunoblotting using anti-centrin antibodies shows that plant centrins vary from 17 to 20 kDa. Immunofluorescence microscopy with anti-centrin antibodies revealed only weak centrin immunoreactivity in the cytoplasm, nucleus, nuclear envelope, phragmoplast and mitotic matrix in meristematic cells. There was a slightly more intense perinuclear labelling in large differentiating onion cells and between separating anaphase chromosomes. While centrin is known to localise to the mitotic spindle poles in animal and algal cells, there was no appreciable immunoreactivity at the spindle poles in higher plants. In contrast, there was an intense immunofluorescence signal with anti-centrin antibodies in the developing cell plate. Further characterisation of the cell plate labelling by immunogold electron microscopy shows centrin immunoreactivity was closely associated with vesicles in the cell plate. Our observations suggest that centrin may play a role in cell plate formation.Abbreviations BSA
bovine serum albumin
- MTs
microtubules
- MTOCs
microtubule organising centres
- PBS
phosphate buffered saline
- PBST
phosphate buffered saline with Tween-20 相似文献
22.
Nichollas E Scott Lindsay D Rogers Anna Prudova Nat F Brown Nikolaus Fortelny Christopher M Overall Leonard J Foster 《Molecular systems biology》2017,13(1)
Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases. 相似文献
23.
The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP.MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2. 相似文献
24.
Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM. 相似文献
25.
Brecknock S Dibbayawan TP Vesk M Vesk PA Faulkner C Barton DA Overall RL 《Planta》2011,234(4):749-758
Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called
plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport
into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural
components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution
scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher
plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters.
Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were
observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose
or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other
particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD. 相似文献
26.
Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo 总被引:1,自引:0,他引:1
Jefferson T Čaušević M auf dem Keller U Schilling O Isbert S Geyer R Maier W Tschickardt S Jumpertz T Weggen S Bond JS Overall CM Pietrzik CU Becker-Pauly C 《The Journal of biological chemistry》2011,286(31):27741-27750
Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing. 相似文献
27.
Morrison C Mancini S Cipollone J Kappelhoff R Roskelley C Overall C 《The Journal of biological chemistry》2011,286(39):34271-34285
Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix. CLIP-CHIP® microarrays identified changes in the complete protease and inhibitor expression profile of the breast cancer and osteoblast cells that were induced upon co-culture. A large increase in osteoblast-derived MMP-13 mRNA and protein was observed. Affymetrix analysis and validation showed induction of MMP-13 was initiated by soluble factors produced by the breast tumor cells, including oncostatin M and the acute response apolipoprotein SAA3. Significant changes in the osteoblast secretomes upon addition of MMP-13 were identified by degradomics from which six novel MMP-13 substrates with the potential to functionally impact breast cancer metastasis to bone were identified and validated. These included inactivation of the chemokines CCL2 and CCL7, activation of platelet-derived growth factor-C, and cleavage of SAA3, osteoprotegerin, CutA, and antithrombin III. Hence, the influence of breast cancer metastases on the bone microenvironment that is executed via the induction of osteoblast MMP-13 with the potential to enhance metastases growth by generating a microenvironmental amplifying feedback loop is revealed. 相似文献
28.
Summary Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm–1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.Abbreviations PBS
phosphate buffered saline
- PMM
protoplast maintenance medium
- DMM
dilute maintenance medium
- MES
2(N-morpholino)ethanesulfonic acid
- TRIS
tris(hydroxymethyl)aminomethane
- ANOVA
analysis of variance 相似文献
29.
30.
This study tested several aspects of a model proposed by Williamson (1990, 1991) in which stresses in plant cell walls, detected by stress-receptive portions of inelastic cellulose microfibrils, orient microtubules via interactions with cell wall-linked transmembrane proteins. Young expanding cells of pea root tips have highly ordered transverse arrays of microtubules oriented perpendicular to the direction of cell expansion. The recovery of these ordered MT arrays after depolymerisation with oryzalin was assessed. It was shown that treating roots with disruptors of microfibril synthesis (2,6-dichlorobenzonitrile and calcofluor white) or the disruption of Arg-Gly-Asp (RGD)-mediated wall-membrane links did not affect the orientation of recovering microtubule arrays. Furthermore, cell wall stresses themselves appeared unnecessary for regeneration of transverse arrays. The relevance of these findings to Williamson's hypothesis is discussed. 相似文献