RESUMO
Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy-based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.
Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Gelsolina/metabolismo , Miosinas/metabolismo , Animais , Humanos , Miosina Tipo II/metabolismo , Ligação Proteica , CoelhosRESUMO
Human serum albumin (HSA) is the most abundant plasma protein in circulation. The three most important drug-binding sites on HSA are Sudlow's Site I (subdomain IIA), Sudlow's Site II (subdomain IIIA), and Heme site (subdomain IB). Heme site and Site I are allosterically coupled; therefore, their ligands may be able to allosterically modulate the binding affinity of each other. In this study, the effects of four Heme site ligands (bilirubin, biliverdin, hemin, and methyl orange) on the interaction of the Site I ligand warfarin with HSA were tested, employing fluorescence spectroscopic, ultrafiltration, and ultracentrifugation studies. Our major results/conclusions are the following. (1) Quenching studies indicated no relevant interaction, while the other fluorescent model used suggested that each Heme site ligand strongly decreases the albumin binding of warfarin. (2) Ultrafiltration and ultracentrifugation studies demonstrated the complex modulation of warfarin-HSA interaction by the different Heme site markers; for example, bilirubin strongly decreased while methyl orange considerably increased the bound fraction of warfarin. (3) Fluorescence spectroscopic studies showed misleading results in these diligand-albumin interactions. (4) Different Heme site ligands can increase or decrease the albumin binding of warfarin and the outcome can even be concentration dependent (e.g., biliverdin and hemin).
Assuntos
Biliverdina , Varfarina , Humanos , Varfarina/farmacologia , Heme/metabolismo , Hemina , Bilirrubina , Ligantes , Albumina Sérica/metabolismoRESUMO
The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Gelsolina/metabolismo , Tropomiosina/química , Citoesqueleto de Actina/química , Gelsolina/química , Humanos , Modelos Moleculares , Polimerização , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Tropomiosina/metabolismoRESUMO
Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Deleção de Genes , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de ProteínaRESUMO
BACKGROUND: Simultaneous determination of the two main actin scavenger proteins in sepsis has not been investigated until now. In our pilot study, we elucidated the predictive values of Gc globulin and gelsolin (GSN) in sepsis by comparing them to classic laboratory and clinical parameters. METHODS: A 5-day follow-up was performed, including 46 septic patients, 28 non-septic patients and 35 outpatients as controls. Serum Gc globulin and GSN levels were determined by automated immune turbidimetric assay on a Cobas 8000/c502 analyzer. Patients were retrospectively categorized according to the sepsis-3 definitions, and 14-day mortality was also investigated. RESULTS: First-day GSN also differentiated sepsis from non-sepsis (AUC: 0.88) similarly to C-reactive protein (AUC: 0.80) but was slightly inferior to procalcitonin (PCT) (AUC: 0.98) with a cutoff value of GSN at 22.29 mg/L (sensitivity: 83.3%; specificity: 86.2%). Only first-day SOFA scores (0.88) and GSN (0.71) distinguished septic survivors from non-survivors, whereas lactate (0.99), Gc globulin (0.76) and mean arterial pressure (MAP) (0.74) discriminated septic shock from sepsis. Logistic regression analyses revealed SOFA scores and GSN being significant factors regarding 14-day mortality. First-day GSN levels were higher (p<0.05) in septic survivors than in non-survivors. Gc globulin levels remained higher (p<0.01) in sepsis when compared with septic shock during the follow-up period. CONCLUSIONS: Both serum GSN and Gc globulin may have predictive values in sepsis. Considering the small sample size of our study, further measurements are needed to evaluate our results. Measurement of Gc globulin and GSN maybe useful in assessment of sepsis severity and in therapeutic decision-making.
Assuntos
Gelsolina/sangue , Sepse/diagnóstico , Proteína de Ligação a Vitamina D/sangue , Idoso , Feminino , Humanos , Imunoturbidimetria , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prognóstico , Sepse/sangueRESUMO
BACKGROUND: Studies showing the potential predictive value of the actin-binding protein gelsolin, in critically ill patients are scarce. Moreover, even up to now a rapid automated measurement of gelsolin has still remained a challenge. Therefore, we developed and validated an automated serum gelsolin immune turbidimetric assay for possible clinical use. METHODS: Validation of serum gelsolin assay was performed on a Cobas 8000/c502 analyzer (Roche) according to the second edition of Eurachem guidelines. Furthermore, we also studied the diagnostic value of serum gelsolin in sepsis when investigating sera of septic (n = 25), systemic inflammatory response syndrome (SIRS; n = 8) and control patients (n = 14). We compared our previously published Western blot data with those of the new turbidimetric assay. RESULTS: The sample volume was 7 µL and the assay time was 10 minutes. The detection limit was 0.72 mg/L, intra- and inter-assay imprecision remained in most cases less than 5% expressed as CV. Recovery was found to be 84.56%-93.52% and linearity study gave an appropriate correlation coefficient by linear regression analysis (r2 = .998). Septic patients exhibited lower (P = .015) first-day serum gelsolin levels than SIRS patients, which confirmed our previous Western blot results. The determined cut-off point for serum gelsolin was 14.05 mg/L (sensitivity: 75%; specificity: 60%) when investigating its diagnostic value in sepsis. CONCLUSION: Based on the results, our immune turbidimetric measurement offers a rapid and accurate quantitation of gelsolin in human serum samples. Serum gelsolin seems a promising additional diagnostic marker of sepsis which has to be further investigated.
Assuntos
Automação Laboratorial/métodos , Gelsolina/sangue , Nefelometria e Turbidimetria/métodos , Sepse/sangue , Sepse/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Curva ROC , Reprodutibilidade dos TestesRESUMO
The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Musculares/química , Proteínas Quinases/química , Sequência de Aminoácidos , Conectina , Guanidina/farmacologia , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Concentração Osmolar , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TemperaturaRESUMO
Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1-3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4-6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (â¼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (â¼µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4-6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1-3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4-6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.
RESUMO
Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.
Assuntos
Actinas/química , Proteínas Musculares/química , Proteínas Quinases/química , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Biofísica , Conectina , Humanos , Técnicas In Vitro , Contração Muscular/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Nanotecnologia , Pinças Ópticas , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , ViscosidadeRESUMO
Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy. The magnitude of these forces provided a direct measure of cell stiffness, cell membrane effective surface viscosity and association with the underlying cytoskeleton. Furthermore, we measured the lateral diffusion constant of a lipid analog DiIC12, using fluorescence recovery after photobleaching, which offers additional information on the organization of the membrane. We found that cholesterol depletion significantly increased the adhesion energy between the membrane and the cytoskeleton and decreased the membrane diffusion constant. An increase in cellular cholesterol to a level higher than that in control cells led to a decrease in the adhesion energy and the membrane surface viscosity. Disassembly of the actin network abrogated all the observed effects, suggesting that cholesterol affects the mechanical properties of a cell through the underlying cytoskeleton. The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis.
Assuntos
Membrana Celular/química , Colesterol/química , Citoesqueleto/química , Células Endoteliais/química , Animais , Aterosclerose , Fenômenos Biomecânicos , Carbocianinas/química , Bovinos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Colesterol/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Microscopia de Força Atômica , Microscopia de Fluorescência , Nanotubos/química , FotodegradaçãoRESUMO
The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.