Iago Molist - Academia.edu (original) (raw)

Papers by Iago Molist

(A) The indicated strains INN1-GFP C2-HOF1 (YIMP19... more (A) The indicated strains INN1-GFP C2-HOF1 (YIMP196) and INN1-GFP td-cyk3-aid C2-HOF1 (YIMP198) were released from G1 arrest at 24°C in YPRaff medium and cells were allowed to progress through the cell cycle at 24°C in YPGal after depleting Td-Cyk3-aid. The proportion of binucleate cells was monitored (i) in parallel with recruitment of Inn1 to the bud-neck (ii). Examples of cells with Inn1-GFP rings at the bud-neck are shown for the 105’ time-point. Scale bars indicate 2μm (iii). (B) The indicated strains INN1-GFP C2-HOF1 td-cyk3-aid CYK3 (YMF951) and INN1-GFP C2-HOF1 td-cyk3-aid cyk3-2A (YMF950) were grown as in (A). Recruitment of Inn1 to the bud-neck was determined. (C) The indicated strains from Fig 9A INN1-GFP C2-HOF1 (YIMP196) and INN1-GFP td-cyk3-aid C2-HOF1 (YIMP198) were released from G1 arrest at 24°C in YPRaff medium after depletion of Td-Cyk3-aid. After cells budded and completed S-phase, nocodazole was added to synchronise the cells in G2-M-phase. The recruitment of Inn1 to the bud-neck in cells arrested in G2-M phase was monitored and examples of cells with Inn1-GFP rings at the bud-neck in nocodazole-arrested cells are shown. Scale bars indicate 2μm. (D) Control (YIMP234) and td-cyk3-aid C2-HOF1 (YIMP246) strains were grown as described in (A) but calcofluor was added upon release from G1 block. The proportion of binucleate cells was determined (i) and the number of cells forming primary septa stained with calcofluor was counted (ii). Examples of calcofluor-stained cells from 135 minutes after release from G1 block (iii). Scale bars correspond to 2μm.</p

(A) Truncated allele of Chs2 lacking transmembrane domain (Chs2-1-629... more (A) Truncated allele of Chs2 lacking transmembrane domain (Chs2-1-629) and fragment containing the tail of Chs2 (1–215) interact in a yeast two-hybrid assay with C-terminus of Inn1 (134–409). (B) Control cells (YAD382) and INN1-C-terminus-TAP cells (YMF82) were grown at 24°C in YPD medium, arrested in G1 phase and released for 105 minutes. Inn1 C-terminus-TAP was immunoprecipitated from cell extracts on IgG beads before the detection of the indicated proteins by immunoblotting. (C) C-terminus of Inn1 inhibits the catalytic activity of Chs2. The protein levels of overexpressed Chs2 and C-terminus (i) and percentage of active chitin synthase (ii) in control and cells lacking Chs3 and overexpressing either GAL-CHS2 (YMF687) or GAL-CHS2 GAL-Cterminus (YMF673) were determined in membranes isolated from asynchronous cultures (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a>). (D) The protein levels of overexpressed Chs2 and Inn1 (i) and percentage of active chitin synthase (ii) in control and cells lacking Chs3 and overexpressing either GAL-CHS2 (YMF687) or GAL-CHS2 GAL-INN1 (YMF561) were determined in membranes isolated from asynchronous cultures (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a>).</p

(A) Summary of yeast two-hybrid interactions between the fragment of ... more (A) Summary of yeast two-hybrid interactions between the fragment of Chs2 containing the catalytic domain (Chs2-215-629) and fragments of Cyk3. (B) Full-length Cyk3 interacts directly with Chs2-215-629. Pairs of E. coli cell cultures expressing 6His-tagged-Cyk3 and Strep-tag-Chs2-215-629 were mixed and used to purify putative protein complexes via Strep-Tactin Superflow and Ni-NTA agarose resins, following the scheme depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#pgen.1005864.s001" target="_blank">S1A Fig</a>. The final purified fractions were analysed by SDS-PAGE and the tagged proteins were detected with anti-Streptag or anti-His antibodies. (C) Inn1, Cyk3 and the catalytic domain of Chs2 form a stable complex. Experiment was performed in a similar way to what is presented in (B) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a>). Cultures were mixed so that the cell extract would contain three recombinant proteins (6His-tagged-Inn1, Strep-tag-Chs2-215-629 and untagged Cyk3). In the case of controls, a culture with an empty vector was mixed with the corresponding cultures expressing the other two recombinant proteins. Chs2 and Inn1 tagged proteins were detected with anti-Streptag or anti-His antibodies respectively, and Cyk3 protein was detected with anti-Cyk3 antibodies.</p

(A) Tetrad analysis of the meiotic progeny from the indicated diploid... more (A) Tetrad analysis of the meiotic progeny from the indicated diploid strain (YIMP11) shows that CHS2-V377I allows C2-HOF1 cyk3Δ cells to grow. Spores of the indicated genotypes were grown for 24 hours on YPD plates at 24°C. Scale bars correspond to 20μm. (B) Serial dilutions of strains YJW15 (1), YIMP60 (2), YIMP43 (3), YIMP41 (4), YIMP149 (5), YIMP147 (6) and YIMP142 (7) were plated on YPD medium or YPD medium containing auxin and incubated for three days at 24°C. Functional domains or proteins under restrictive conditions are indicated. (C) Tetrad analysis of the meiotic progeny from diploid strain (YIMP12) shows that C2-K31A-HOF1 cyk3Δ cells are unable to grow. Defects can be rescued by the expression of hypermorphic allele CHS2-V377I. Scale bars correspond to 20μm. (D) Tetrad analysis of the meiotic progeny from diploid strain (YIMP388) shows that the Inn1 C-terminus effect and the lack of Cyk3 can be fully bypassed by hypermorphic CHS2 (CHS2-V377I). (E) Tetrad analysis of the meiotic progeny from diploid strain (YIMP437) shows that C2-HOF1 inn1Δ cyk3-2A cells are unable to grow and defects can be rescued by the expression of hypermorphic allele CHS2-V377I.</p

(A) The indicated strains CHS2-GFP (YMF330) and &l... more (A) The indicated strains CHS2-GFP (YMF330) and CHS2-GFP iqg1-td (YMF305) were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 37°C to deplete Iqg1-td. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings or spots of Chs2 at the cleavage site (ii). Examples of cells with Chs2-GFP rings at the bud-neck are shown for the 75’ time-point (iii). (B) The indicated strains HOF1-GFP (YASD550) and HOF1-GFP iqg1-td (YASD556) were grown as in (A). The proportion of binucleate cells (i) and the recruitment of Hof1 to the site of division (ii) were monitored. Examples of cells with Hof1-GFP rings at the bud-neck are shown for the 75’ time-point (iii). (C) The indicated strains CHS2-GFP (YMF330) and CHS2-GFP inn1-td hof1-td (YIMP230) were grown as in (A). The proportion of binucleate cells was studied (i) together with Chs2 localisation at the cleavage site (ii). Examples of cells with Chs2-GFP rings at the bud-neck are shown for the 75’ time-point (iii). Scale bars correspond to 2μm.</p

(A) Truncated allele of Chs2 containing the catalytic domain and Inn1... more (A) Truncated allele of Chs2 containing the catalytic domain and Inn1 were used to show that this region of Chs2 (Chs2-215-629) interacts in a yeast two-hybrid assay with Inn1. (B) Chs2-215-629 interacts directly with full-length Inn1. After induction with IPTG, pairs of E. coli cell cultures expressing the indicated recombinant protein fragments were mixed and used to purify putative protein complexes. The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie (i). Both Inn1 and Chs2-215-629 migrate similarly in SDS-PAGE gels (6His-Inn1(49kDa) and Strep-tag-Chs2-215-629 (51kDa)). The acrylamide band was cut out from the gel and protein composition, spectral counts and coverage were analysed by mass spectrometry (ii). The final purified fractions from (i) were analysed by immunoblotting and the tagged proteins were detected with anti-Streptag or anti-His antibodies (iii). (C) Chs2-1-629 interacts directly with full-length Inn1. Pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify and analyse putative protein complexes as in (B). (D) After induction with IPTG (i) pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify and analyse putative protein complexes as in (B) (ii).</p

(A)INN1-TAP CHS2-9MYC (YMF38), C2-TAP CHS... more (A)INN1-TAP CHS2-9MYC (YMF38), C2-TAP CHS2-9MYC (YMF88) and control (YAD382) strains were grown at 24°C in YPD medium, arrested in G1 phase by the addition of alpha factor, and then released in YPD medium for 105 minutes. Cell extracts were made and Inn1-TAP or C2-TAP were immunoprecipitated on IgG beads before detection of the indicated proteins by immunoblotting (i). After induction with IPTG, pairs of E. coli cultures expressing 6His-tagged-Inn1-C2 and Strep-tag-Chs2-215-629 were mixed and used to purify putative protein complexes following scheme in S1A (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a>). The final purified fractions were analysed by SDS-PAGE and tagged proteins were detected with anti-Streptag or anti-His antibodies (ii). Pairs of E. coli cultures expressing 6His-tagged-Inn1-C2 or 6His-tagged-Inn1-C2-K31A were mixed with Strep-tag-Chs2-215-629 and used to purify putative protein complexes as in (ii). The final purified fractions were analysed by SDS-PAGE and tagged proteins were detected with anti-Streptag or anti-His antibodies (iii). (B) chs3Δ control (YMF505) and GAL-C2 chs3Δ (YRK3) cells were grown in YPRaff medium at 24°C and synchronised in G1 with alpha factor. Subsequently, cells were released in YPGal for 135 minutes from G1 block in the presence of calcofluor to visualise primary septum deposition. 100 cells with primary septum for each sample were examined and we found that 15% of GAL-C2 chs3Δ cells had clearly higher intensity at the primary septum than the average intensity in control cells. Examples of these cells are shown in (i). Scale bars correspond to 2μm. The relative signal intensity of primary septum was measured for 100 cells and compared to control cells, where signal intensity was set to 100% (ii). (C) C2 domain of Inn1 increases the catalytic activity of Chs2. The protein levels of overexpressed Chs2 and C2 proteins (i) and percentage of active chitin synthase (ii) in chs3Δ control cells and cells lacking Chs3 and overexpressing either GAL-CHS2 (YMF687) or GAL-CHS2 GAL-C2 (YMF581) were determined in membranes isolated from asynchronous cultures (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a>). Control, GAL-CHS2 (YMF687) and GAL-CHS2 GAL-C2 (YMF581) were grown as in (B) and stained with calcofluor to visualise primary septum deposition. 100 cells with primary septum for each sample were examined and examples of these cells are shown in (iii) and the relative signal intensity of primary septum was measured and compared to control cells, where signal intensity was set to 100% (iv). Scale bars correspond to 2μm. (D) The chitin synthase activity in chs3Δ cells expressing CHS2 (YMF191), C2-CHS2 (YMF172), C2-K31A-CHS2 (YMF174) or CHS2-V377I (YMF192) was determined as in (C) (i). Cells were grown in YPD containing 0.1mM CuSO4 since CHS2 and CHS2 fusions were under the control of the CUP1 promoter and protein expression levels of Chs2 and its fusions were determined (ii). Note that CHS2 is highly expressed in (C), under the control of the GAL1-10 promoter, whereas CHS2 levels are much reduced in (D), under the CUP1 promoter control.</p

(A) Tetrad analysis of the meiotic progeny from the indicated diploid... more (A) Tetrad analysis of the meiotic progeny from the indicated diploid strain (YMF669) shows that inactivation of the transglutaminase-like domain of Cyk3 (cyk3-2A allele, which contains two point mutations, D578A and H563A) is lethal when combined with C2-HOF1. Scale bars correspond to 20μm. (B) Control (YMF505), GAL-CYK3 (YIMP235) and GAL-cyk3-2A (YMF576) cells were grown in YPRaff medium at 24°C, synchronised in G1 with alpha factor, and cells were released in YPGal for 135 minutes in the presence of calcofluor to visualise primary septum deposition (i). The relative signal intensity of primary septum was measured for 100 cells and compared to control cells, where signal intensity was set to 100% (ii). Scale bars correspond to 2μm.</p

(A)INN1-TAP CHS2-9MYC (YMF38) and control (YMF79) ... more (A)INN1-TAP CHS2-9MYC (YMF38) and control (YMF79) were grown at 24°C in YPD medium and synchronised in G1 phase with mating pheromone and then released for 105 minutes. Cell extracts were prepared before the immunoprecipitation of Inn1-TAP (or TAP in control) on IgG-beads. The isolated material was released from the beads by cleavage with TEV protease. After cleavage, part of the TAP tag (CBP, calmodulin-binding protein) remained fused to Inn1. Purified material was subjected to immunoprecipitation of Chs2-9MYC before analysis by SDS-PAGE and immunoblotting (i). The protein composition of purified fractions including spectral counts and coverage were analysed by mass spectrometry (ii). The INN1-TAP IQG1-6HA (YMF149) strain together with control (YMF152) were grown as in (i) and cell extracts were prepared and analysed by SDS-PAGE and immunoblotting (iii). (B) IQG1-6HA (YMF152) and control (YAD382) strains were grown as in (A) and cell extracts were prepared before the immunoprecipitation of Iqg1 and detection of the indicated proteins by immunoblotting. (C) MYO1-FLAG (YMF362) strain was grown at 24°C in YPD medium and synchronised in G1 phase with mating pheromone (G1 sample) or released from G1 block for 30 minutes (S-phase sample) or 105 minutes (cytokinesis sample). Cell cycle progression was monitored by flow cytometry (i). Cell extracts were then prepared before the immunoprecipitation of Myo1-FLAG on anti-FLAG-beads and detection of the indicated proteins by immunoblotting (ii).</p

(A) The indicated strains MYO1-GFP C2-HOF1 (YIMP22... more (A) The indicated strains MYO1-GFP C2-HOF1 (YIMP225) and MYO1-GFP td-cyk3-aid C2-HOF1 (YIMP209) were released from G1 arrest at 24°C in YPRaff medium and cells were allowed to progress through the cell cycle at 24°C in YPGal after depleting Td-Cyk3-aid. The proportion of binucleate cells was monitored (i) in parallel with recruitment of Myo1 to the bud-neck (ii). Examples of cells with Myo1-GFP rings at the bud-neck are shown for the 105’ time-point. Scale bars indicate 2μm (iii). (B) The indicated strains MYO1-GFP C2-HOF1 SPC42-EQFP (YIMP452) and MYO1-GFP td-cyk3-aid C2-HOF1 (YIMP209) were grown and arrested in G1 phase with mating pheromone at 24°C in YPRaff and subsequently cells were released from G1 arrest at 24°C in YPGal after depleting Td-Cyk3-aid. After cells budded and completed S-phase, nocodazole was added to synchronise the cells in G2-M-phase. Cells were then washed into fresh Synthetic Complete medium and subsequently cells were placed in the time-lapse slide to examine the localisation of Myo1 every two minutes as cells completed mitosis at 24°C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005864#sec011" target="_blank">Materials and Methods</a> for details). Contraction of the actomyosin ring in control cells ends as a “spot”, whereas in mutant cells, the actomyosin ring disappears before that stage. Examples of cells in which time-lapse analysis was used to follow the contraction of the Myo1-GFP ring. Scale bars indicate 2μm (iii).</p

This study YIMP457 MATa ura3-52 lys2∆201 pep4∆ pDDGFP-CHS2-TEV-GFP-8HIS (URA3) This study YIMP458... more This study YIMP457 MATa ura3-52 lys2∆201 pep4∆ pDDGFP-CHS2-TEV-GFP-8HIS (URA3) This study YIMP458 MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 pep4∆::ADE2 pDDGFP-CHS2-TEV-GFP-8HIS (URA3) This study YIMP459 MATa ura3-52 lys2∆201 pep4∆ pDDGFP-CHS2(215-963)-TEV-GFP-8HIS (URA3) This study YIMP460 MATa ura3-52 lys2∆201 pep4∆ pDDGFP-C2-CHS2-TEV-GFP-8HIS (URA3) This study

Chemical Science, 2017

The critical contribution of membrane proteins in normal cellular function makes their detailed s... more The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human b 2 adrenergic receptor (b 2 AR) and its complex with G s protein. A further evaluation of this DTM led to a clear visualization of b 2 AR-G s complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

Resumen del trabajo presentado a la 10a Reunion de la Red Espanola de Levaduras, celebrada en El ... more Resumen del trabajo presentado a la 10a Reunion de la Red Espanola de Levaduras, celebrada en El Escorial (Madrid) del 16 al 18 de diciembre de 2015.

Organic & biomolecular chemistry, Jan 4, 2018

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecula... more Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred mar...

Resumen del poster presentado al Ramon Areces Foundation International Symposium: Cell Proliferat... more Resumen del poster presentado al Ramon Areces Foundation International Symposium: Cell Proliferation and Genome Integrity, celebrado en Santander (Espana) del 3 al 4 de abril de 2014.

Methods in molecular biology (Clifton, N.J.), 2016

A number of model organisms have provided the basis for our understanding of the eukaryotic cell ... more A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

PLOS Genetics, 2016

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together... more Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named 'ingression progression complexes' (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the coordinated regulation of cell division in budding yeast, in which IPCs play a central role.

Chemico-biological interactions

Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) ... more Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding...

(A) Tetrad analysis of the meiotic progeny from the indicated diploid... more (A) Tetrad analysis of the meiotic progeny from the indicated diploid strain (YMF669) shows that inactivation of the transglutaminase-like domain of Cyk3 (cyk3-2A allele, which contains two point mutations, D578A and H563A) is lethal when combined with C2-HOF1. Scale bars correspond to 20μm. (B) Control (YMF505), GAL-CYK3 (YIMP235) and GAL-cyk3-2A (YMF576) cells were grown in YPRaff medium at 24°C, synchronised in G1 with alpha factor, and cells were released in YPGal for 135 minutes in the presence of calcofluor to visualise primary septum deposition (i). The relative signal intensity of primary septum was measured for 100 cells and compared to control cells, where signal intensity was set to 100% (ii). Scale bars correspond to 2μm.</p

Chemical Science, 2017

Organic & biomolecular chemistry, Jan 4, 2018

Methods in molecular biology (Clifton, N.J.), 2016

PLOS Genetics, 2016

Chemico-biological interactions