Biomedical subjects

M Bezanilla

Publications and source records attributed to M Bezanilla.

14 recordsLinked to original sources

PubMed2005

An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes.

RNAi is a powerful method for generating loss of function mutants, especially for targeting genes belonging to large gene families. We have recently shown that RNAi functions in the moss Physcomitrella patens. We obtained stable lines that show constitutive silencing of a nuclearly localized GFP:GUS fusion protein (NLS:GFP:GUS). However, lines that display silencing of the protein do not necessarily have reduced transcript levels. Therefore, a system has been developed that silences the NLS:GFP:GUS reporter construct at the same time as it silences a gene of interest. Gateway (Invitrogen) recombination cassettes were incorporated into these vectors to facilitate cloning of many different cDNA sequences. In addition, vectors were generated that contain genomic moss DNA sequence information to increase the production of stable moss lines. Transformation with these constructs results in strong silencing within 24 h and is stable for at least a month after transformation. FtsZ2-1, whose loss of function phenotype is known, was incorporated as a test case for analyzing phenotypes. One hundred per cent of regenerating colonies that have silenced GFP exhibit a loss of function FtsZ2-1 phenotype, validating the use of this system to assay phenotypes for plant genes of unknown function.

Blotting, Northern↗

PubMed2000

Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp.

Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects. However, it fails to rescue the Deltamyo1 Deltawsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

Actin-Related Protein 2↗

PubMed2000

Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis.

Myosin-II is required for cytokinesis in Schizosaccharomyces pombe [1-3], but unlike other unicellular organisms, S. pombe has two structurally distinct myosin-IIs, Myo2p and Myp2p, which are required under different conditions [4]. Disruption of myo2(+) is lethal, whereas disruption of myp2(+) leads to defects in cytokinesis when nutrients are limiting and to cold-sensitivity in 1 M KCl. In dividing cells, both myosin-IIs localize to a ring in the center of the cell, which is thought to contract, separating the cytoplasms of the daughter cells. Using deconvolution microscopy, we obtained three-dimensional reconstructions of fission yeast cells expressing green fluorescent protein-labeled (GFP)-myosin-II, providing for the first time detailed images of GFP-myosin-II rings. By time-lapse microscopy, we observed ring assembly and contraction in three dimensions using GFP-tubulin as a cell cycle marker. We determined that the Myo2p ring forms in metaphase/anaphase A whereas the Myp2p ring forms much later, at the end of anaphase B. Myo2p initiates ring formation while Myp2p acts later to increase the efficiency of cytokinesis.

Anaphase↗

PubMed2000

Myosin-II tails confer unique functions in Schizosaccharomyces pombe: characterization of a novel myosin-II tail.

Schizosaccharomyces pombe has two myosin-IIs, Myo2p and Myp2p, which both concentrate in the cleavage furrow during cytokinesis. We studied the phenotype of mutant myosin-II strains to examine whether these myosins have overlapping functions in the cell. myo2(+) is essential. myp2(+) cannot rescue loss of myo2(+) even at elevated levels of expression. myp2(+) is required under specific nutritional conditions; thus myo2(+) cannot rescue under these conditions. Studies with chimeras show that the tails rather than the structurally similar heads determine the gene-specific functions of myp2(+) and myo2(+). The Myo2p tail is a rod-shaped coiled-coil dimer that aggregates in low salt like other myosin-II tails. The Myp2p tail is monomeric in high salt and is insoluble in low salt. Biophysical properties of the full-length Myp2p tail and smaller subdomains indicate that two predicted coiled-coil regions fold back on themselves to form a rod-shaped antiparallel coiled coil. This suggests that Myp2p is the first type II myosin with only one head. The C-terminal two-thirds of Myp2p tail are essential for function in vivo and may interact with components of the salt response pathway.

Animals↗

PubMed1999

Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2.

Targeting of protein modification enzymes is a key biochemical step to achieve specific and effective posttranslational modifications. Two alternatively spliced ZIP1 and ZIP2 proteins are described, which bind to both Kvbeta2 subunits of potassium channel and protein kinase C (PKC) zeta, thereby acting as a physical link in the assembly of PKCzeta-ZIP-potassium channel complexes. ZIP1 and ZIP2 differentially stimulate phosphorylation of Kvbeta2 by PKCzeta. They also interact to form heteromultimers, which allows for a hybrid stimulatory activity to PKCzeta. Finally, ZIP1 and ZIP2 coexist in the same cell type and are elevated differentially by neurotrophic factors. These results provide a mechanism for specificity and regulation of PKCzeta-targeted phosphorylation.

Alternative Splicing↗

PubMed1997

Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis.

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We cloned myp2(+), a myosin-II in Schizosaccharomyces pombe, conditionally required for cytokinesis. myp2(+), the second myosin-II identified in S. pombe, does not completely overlap in function with myo2(+). The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2(+) is not lethal but under nutrient limiting conditions cells lacking myp2(+) function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally, Deltamyp2 cells are cold sensitive in high salt, failing to form colonies at 17 degrees C. Thus, myp2(+) is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Deltamyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Deltamyp2 and three alleles of cdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicating myp2(+) function for efficient cytokinesis under normal conditions.

Actins↗

PubMed1995

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.

Bacteriophage lambda↗

PubMed1994

Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope.

The capability of the scanning force microscope (SFM) to image molecules in aqueous buffers has opened the exciting possibility of following processes of molecular assembly in real time and in near-physiological environments. This capability is demonstrated in this paper by following the assembly process of RNA polymerase-DNA complexes. DNA fragments deposited on mica and imaged in Hepes/MgCl2 are shown before and after Escherichia coli RNA polymerase holoenzyme is injected in the SFM liquid chamber. The protein can recognize and bind to these DNA fragments within several seconds after injection, suggesting that the protein and the DNA retain their native configuration after deposition and during SFM imaging. A time-lapse sequence depicting the process of assembly of RNA polymerase-DNA complexes is shown. These results represent the first step for acquiring the capabilities to monitor complex biomolecular processes as they take place in ionic solutions and to characterize their spatial organization.

Bacterial Proteins↗

PubMed1994

Bending and straightening of DNA induced by the same ligand: characterization with the atomic force microscope.

A new ligand, MGT-6b, binds to DNA with two linked parts: a polyamine that binds to the phosphate backbone and a tripyrrole peptide that binds to the minor groove. This ligand decreases the curvature of bent kinetoplast DNA (kDNA) and also increases the curvature of a 400-bp DNA that is used as a molecular weight standard (M400), as characterized with the atomic force microscope (AFM). MGT-6b is more effective than distamycin at straightening kDNA. MGT-6b retards the electrophoretic mobility of M400 DNA, although neither of its two component parts alters the mobility of M400 or its curvature as seen in the AFM. Thus, both parts of the ligand are needed for the "vise grip" mode of binding that affects DNA bending. the ability of MGT-6b to both bend and straighten DNA is probably related to the very different DNA sequences and, hence, structures of kDNA and M400 DNA.

Bacteriophage phi X 174↗

PubMed1994

Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.

Adsorption↗

PubMed1994

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy.

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2-4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13-16 nm. Monomers found in the absence of ATP show heights of 5-7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.

Antigens, Viral, Tumor↗

PubMed1993

Atomic force microscopy of biochemically tagged DNA.

Small fragments of DNA of known length were made with the polymerase chain reaction. These fragments had biotin molecules covalently attached at their ends. They were subsequently labeled with a chimeric protein fusion between streptavidin and two immunoglobulin G-binding domains of staphylococcal protein A. This tetrameric species was expected to bind up to four DNA molecules via their attached biotin moieties. The DNA-protein complex was deposited on mica and imaged with an atomic force microscope. The images revealed the protein chimera at the expected location at the ends of the strands of DNA as well as the expected dimers, trimers, and tetramers of DNA bound to a single protein.

Bacterial Proteins↗

PubMed1993

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.

1-Propanol↗

PubMedDate not supplied

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air. Finally, AFM images are presented of single-stranded phi X-174 virion DNA with the gene 32 single-stranded binding protein. A summary of the current state of the field and of the methods for preparing and imaging DNA in the AFM is also presented.

Bacteriophage T4↗