Maria João Sarmento | Universidade de Lisboa (original) (raw)
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Papers by Maria João Sarmento
Biochemical Society Transactions, 2014
Ca(2+) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associate... more Ca(2+) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P2 has been shown to be tightly regulated and dependent on the presence of Ca(2+), with co-operative binding in some cases. In these proteins, PI(4,5)P2 and Ca(2+) binding typically occurs at different binding sites. In addition, several PI(4,5)P2-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P2 for protein interaction, translating Ca(2+) transient microdomains into variations of PI(4,5)P2 lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P2-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P2.
Biophysical Journal, 2015
Biophysical Journal, 2012
Biophysical Journal, 2013
Single molecule fluorescence spectroscopy provides the unique possibility to study enzymatic reac... more Single molecule fluorescence spectroscopy provides the unique possibility to study enzymatic reactions with single turnover resolution. using fluorogenic enzyme substrates, the time sequence of enzymatic turnovers can be followed and fluctuations in the turnover rate can be detected. To investigate the possibility of constructing kinetic schemes, we have studied the pH-dependent activity of the enzyme alpha-chymotrypsin. An accurate construction of kinetic schemes is so far limited, however, by the design of most fluorogenic substrates that carry two enzyme-cleavable bonds per fluorophore. using a typical double-substituted Rhodamine 110-based substrate, the reaction proceeds in two steps involving a mono-substituted intermediate that possesses only 30% of the quantum yield of Rhodamine 110. Despite its low brightness, we are able to specifically detect the intermediate based on its different fluorescence lifetime. At high substrate concentrations the only product formed is the intermediate and a simplified but accurate kinetic scheme can be obtained. Our results show pH-dependent activity of alphachymotrypsin but clearly do NOT support stretched exponential kinetics, frequently interpreted as dynamic disorder. We hypothesize that ''stretched exponentials'' might be data analysis artefacts occurring for low signal-to-noise data.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014
Calcium has been shown to induce clustering of PI(4,5)P 2 at high and non-physiological concentra... more Calcium has been shown to induce clustering of PI(4,5)P 2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P 2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P 2 (TopFluor(TF)-PI(4,5)P 2 ), we show that Ca 2+ promotes PI(4,5)P 2 clustering in lipid bilayers at physiological concentrations of both Ca 2+ and PI(4,5)P 2 . Fluorescence depolarization data of TF-PI(4,5)P 2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P 2 and calcium, the average cluster size comprises~15 PI(4,5)P 2 molecules. The presence of Ca 2+ -induced PI(4,5)P 2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P 2 clustering was more pronounced in liquid ordered (l o ) membranes, and the PI(4,5)P 2 -Ca 2+ clusters presented an increased affinity for l o domains. In this way, PI(4,5)P 2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P 2 clustering on l o domains might provide targeted nucleation sites for PI(4,5)P 2 clusters upon calcium stimulus.
Characterization of phase coexistence in biologically relevant lipid mixtures is often carried ou... more Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.
Biochemical Society Transactions, 2014
Ca(2+) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associate... more Ca(2+) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P2 has been shown to be tightly regulated and dependent on the presence of Ca(2+), with co-operative binding in some cases. In these proteins, PI(4,5)P2 and Ca(2+) binding typically occurs at different binding sites. In addition, several PI(4,5)P2-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P2 for protein interaction, translating Ca(2+) transient microdomains into variations of PI(4,5)P2 lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P2-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P2.
Biophysical Journal, 2015
Biophysical Journal, 2012
Biophysical Journal, 2013
Single molecule fluorescence spectroscopy provides the unique possibility to study enzymatic reac... more Single molecule fluorescence spectroscopy provides the unique possibility to study enzymatic reactions with single turnover resolution. using fluorogenic enzyme substrates, the time sequence of enzymatic turnovers can be followed and fluctuations in the turnover rate can be detected. To investigate the possibility of constructing kinetic schemes, we have studied the pH-dependent activity of the enzyme alpha-chymotrypsin. An accurate construction of kinetic schemes is so far limited, however, by the design of most fluorogenic substrates that carry two enzyme-cleavable bonds per fluorophore. using a typical double-substituted Rhodamine 110-based substrate, the reaction proceeds in two steps involving a mono-substituted intermediate that possesses only 30% of the quantum yield of Rhodamine 110. Despite its low brightness, we are able to specifically detect the intermediate based on its different fluorescence lifetime. At high substrate concentrations the only product formed is the intermediate and a simplified but accurate kinetic scheme can be obtained. Our results show pH-dependent activity of alphachymotrypsin but clearly do NOT support stretched exponential kinetics, frequently interpreted as dynamic disorder. We hypothesize that ''stretched exponentials'' might be data analysis artefacts occurring for low signal-to-noise data.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014
Calcium has been shown to induce clustering of PI(4,5)P 2 at high and non-physiological concentra... more Calcium has been shown to induce clustering of PI(4,5)P 2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P 2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P 2 (TopFluor(TF)-PI(4,5)P 2 ), we show that Ca 2+ promotes PI(4,5)P 2 clustering in lipid bilayers at physiological concentrations of both Ca 2+ and PI(4,5)P 2 . Fluorescence depolarization data of TF-PI(4,5)P 2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P 2 and calcium, the average cluster size comprises~15 PI(4,5)P 2 molecules. The presence of Ca 2+ -induced PI(4,5)P 2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P 2 clustering was more pronounced in liquid ordered (l o ) membranes, and the PI(4,5)P 2 -Ca 2+ clusters presented an increased affinity for l o domains. In this way, PI(4,5)P 2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P 2 clustering on l o domains might provide targeted nucleation sites for PI(4,5)P 2 clusters upon calcium stimulus.
Characterization of phase coexistence in biologically relevant lipid mixtures is often carried ou... more Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.