RESUMO
Mitochondrial single-stranded DNA-binding protein (mtSSB) is essential for mitochondrial DNA (mtDNA) replication. Recently, several mtSSB variants have been associated with autosomal dominant mitochondrial optic atrophy and retinal dystrophy. Here, we have studied at the molecular level the functional consequences of one of the most severe mtSSB variants, R107Q. We first studied the oligomeric state of this variant and observed that the mtSSBR107Q mutant forms stable tetramers in vitro. On the other hand, we showed, using complementary single-molecule approaches, that mtSSBR107Q displays a lower intramolecular ssDNA compaction ability and a higher ssDNA dissociation rate than the WT protein. Real-time competition experiments for ssDNA-binding showed a marked advantage of mtSSBWT over mtSSBR107Q. Combined, these results show that the R107Q mutation significantly impaired the ssDNA-binding and compacting ability of mtSSB, likely by weakening mtSSB ssDNA wrapping efficiency. These features are in line with our molecular modeling of ssDNA on mtSSB showing that the R107Q mutation may destabilize local interactions and results in an electronegative spot that interrupts an ssDNA-interacting-electropositive patch, thus reducing the potential mtSSB-ssDNA interaction sites.
Assuntos
DNA de Cadeia Simples , Proteínas de Ligação a DNA , Mutação , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/química , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
We experimentally probe nonlinear wave propagation in weakly compressed granular media and observe a crossover from quasilinear sound waves at low impact to shock waves at high impact. We show that this crossover impact grows with the confining pressure P0, whereas the shock wave speed is independent of P0-two hallmarks of granular shocks predicted recently. The shocks exhibit surprising power law attenuation, which we model with a logarithmic law implying that shock dissipation is weak and qualitatively different from other granular dissipation mechanisms. We show that elastic and potential energy balance in the leading part of the shocks.
RESUMO
Mitochondrial transcription factor A (TFAM) plays a key role in the organization and compaction of the mitochondrial genome. However, there are only a few simple and accessible methods available to observe and quantify TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS) is a straightforward single-molecule force spectroscopy technique. It allows one to track many individual protein-DNA complexes in parallel and to quantify their mechanical properties. Total internal reflection fluorescence (TIRF) microscopy is a high-throughput single-molecule technique that permits the real-time visualization of the dynamics of TFAM on DNA, parameters inaccessible with classical biochemistry tools. Here we describe, in detail, how to set up, perform, and analyze AFS and TIRF measurements to study DNA compaction by TFAM.
Assuntos
DNA , Fenômenos Mecânicos , DNA/química , Proteínas Mitocondriais/genética , Microscopia de Fluorescência/métodos , Análise Espectral/métodos , Acústica , DNA Mitocondrial/genéticaRESUMO
The 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and in the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mitochondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6- and NUDT9-deficiency and found that the depletion of either of the two proteins results in an increased activity of the respiratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD+(H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR.
Assuntos
NAD , Pirofosfatases , Humanos , Hidrólise , Mitocôndrias/metabolismo , Pirofosfatases/genética , Pirofosfatases/química , Pirofosfatases/metabolismoRESUMO
Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression, and packaging of mitochondrial DNA (mtDNA). Recently, a pathogenic homozygous variant in TFAM (P178L) has been associated with a severe mtDNA depletion syndrome leading to neonatal liver failure and early death. We have performed a biochemical characterization of the TFAM variant P178L in order to understand the molecular basis for the pathogenicity of this mutation. We observe no effects on DNA binding, and compaction of DNA is only mildly affected by the P178L amino acid change. Instead, the mutation severely impairs mtDNA transcription initiation at the mitochondrial heavy and light strand promoters. Molecular modeling suggests that the P178L mutation affects promoter sequence recognition and the interaction between TFAM and the tether helix of POLRMT, thus explaining transcription initiation deficiency.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Multimerização Proteica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/ultraestrutura , Animais , Células COS , Chlorocebus aethiops , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Schizosaccharomyces/metabolismoRESUMO
We report on direct measurements of the basal force components for granular material flowing down a smooth incline. We investigate granular flows for a large range of inclination angles from θ=13.4^{∘} to 83.6° and various gate openings of the chute. We find that the effective basal friction coefficient µ_{B}, obtained from the ratio of the longitudinal force to the normal one, exhibits a systematic increase with increasing slope angle and a significant weakening with increasing particle holdup H (the depth-integrated particle volume fraction). At low angles, the basal friction is slightly less than or equal to tanθ. The deviation from tanθ can be interpreted as a contribution from the sidewall to the overall friction. At larger angles, the basal friction µ_{B} saturates at an asymptotic value that is dependent on the gate opening of the chute. Importantly, our data confirm the outcomes of recent discrete numerical simulations. First, for steady and fully developed flows as well as for moderately accelerated ones, the variation of the basal friction can be captured through a unique dimensionless number, the Froude number Fr, defined as Fr=U[over ¯]/(gHcosθ)^{1/2}, where U[over ¯] is the mean flow velocity. Second, the mean velocity scales with the particle holdup H with a power exponent close to 1/4, contrasting with the Bagnold scaling (U[over ¯]â¼H^{3/2}).
RESUMO
The molecular motor protein Kinesin-1 drives intracellular transport of vesicles, by binding to microtubules and making hundreds of consecutive 8-nm steps along them. Three important parameters define the motility of such a linear motor: velocity, run length (the average distance traveled), and the randomness (a measure of the stochasticity of stepping). We used total internal reflection fluorescence microscopy to measure these parameters under conditions without external load acting on the motor. First, we tracked the motility of single motor proteins at different adenosine triphosphate (ATP) concentrations and determined both velocity and (for the first time, to our knowledge, by using single-molecule fluorescence assays) randomness. We show that the rate of Kinesin-1 at zero load is limited by two or more exponentially distributed processes at high ATP concentrations, but that an additional, ATP-dependent process becomes the sole rate-limiting process at low ATP concentrations. Next, we measured the density profile of moving Kinesin-1 along a microtubule. This allowed us to determine the average run length in a new way, without the need to resolve single-molecules and to correct for photobleaching. At saturating ATP concentration, we measured a run length of 1070 +/- 30 nm. This value did not significantly change for different ATP concentrations.
Assuntos
Cinesinas/química , Trifosfato de Adenosina/química , Algoritmos , Animais , Humanos , Cinética , Microscopia de Fluorescência/métodos , Microtúbulos/química , Modelos Químicos , Movimento (Física) , Fotodegradação , Processos Estocásticos , SuínosRESUMO
One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.
Assuntos
Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Fluorescência , Corantes Fluorescentes/químicaRESUMO
The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.
Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , DNA Mitocondrial/química , DNA Viral/química , DNA Viral/genética , Humanos , Ligação Proteica , Multimerização Proteica , Fatores de Transcrição/químicaRESUMO
Mitochondria organize their genome in protein-DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.
Assuntos
DNA Mitocondrial/química , Proteínas de Ligação a DNA/química , Proteínas Mitocondriais/química , Fatores de Transcrição/química , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Desnaturação de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches are addressed, we first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.
Assuntos
Fluorescência , Microscopia de Fluorescência/métodos , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Corantes FluorescentesRESUMO
Lateral diffusion of proteins in the plane of a biological membrane is important for many vital processes, including energy conversion, signaling, chemotaxis, cell division, protein insertion, and secretion. In bacteria, all these functions are located in a single membrane. Therefore, quantitative measurements of protein diffusion in bacterial membranes can provide insight into many important processes. Diffusion of membrane proteins in eukaryotes has been studied in detail using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single-molecule fluorescence (SMF) microscopy. In case of bacteria, such experiments are intrinsically difficult due to the small size of the cells. Here, we review these experimental approaches to quantify diffusion in general and their strengths and weaknesses when applied to bacteria. In addition, we propose a method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs). We demonstrate the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins. Using computer simulations, we study the effect of cell dimensions and membrane curvature on measured CPDs. We find that at least two mobile populations with distinct diffusion coefficients (of 7 and 169 nm(2) ms(-1) , respectively) are necessary to explain the experimental data. The approach described here should be widely applicable for the quantification of membrane-protein diffusion in living bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Simulação por Computador , Difusão , Proteínas de Escherichia coli/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência/métodos , Modelos Biológicos , Espectrometria de Fluorescência/métodosRESUMO
The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs). Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs, but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs, displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F's crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation.