RESUMO
The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.
Assuntos
Citoesqueleto/metabolismo , Filamentos Intermediários/metabolismo , Polímeros/metabolismo , Vimentina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Forma Celular , Cricetinae , Citoesqueleto/ultraestrutura , Filamentos Intermediários/ultraestrutura , Microscopia Confocal , Microtúbulos/metabolismo , Estresse MecânicoRESUMO
The growth of filamentous fungi is a complex process that involves hyphal elongation and branching. Microscopic observations provide a wealth of information on fungal growth, although often requiring laborious manual intervention to record and analyze images. Here, we introduce a novel tool for automated tracking of growth in fungal hyphae that affords quantitative analysis of growth rate and morphology. We supplied a student-grade bright field microscope with stepper motors to enable computer-control of the microscope stage. In addition, we developed an image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates. To achieve continuous observation of hyphal growth, our system automatically maintains the observed sample within field-of-view and performs periodic autofocus correction in the microscope. We demonstrate automated, continuous tracking of hyphal growth in Trichoderma atroviride with sampling rates of seconds and observation times of up to 14â¯h. Tracking records allowed us to determine that T. atroviride hyphae grow with characteristic elongation rates of â¼70â¯nm/s. Surprisingly, we found that prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. These arrest events presented occasionally for subapical branching as well. Finally, from tracking data we found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362⯵m. Altogether, these results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics.
Assuntos
Fungos/ultraestrutura , Hifas/crescimento & desenvolvimento , Microscopia de Vídeo , Morfogênese , Citoesqueleto/ultraestrutura , Fungos/crescimento & desenvolvimento , Hifas/ultraestruturaRESUMO
This study evaluated the aqueous extraction of galactomannans from the seeds of Mimosa scabrella (GM), Stryphnodendron adstringens (GS) and Schizolobium parahybae (GG) for 1, 2, 3, 4, 6, 24 and 48 h. The efficiency of extraction processes was assessed in terms of yield, carbohydrate and protein content. The extraction process, as well as the source of the galactomananns generated molecules with differences in molar mass, viscosity and rigidity analyzed by HPSEC-MALLS/RI/VIS. The extraction time results for each species, based on minimum extraction time and HPSEC-MALLS/RI/VIS results, were 4 h (GM4h), 6 h (GS6h) and 2 h (GG2h) for GM, GS and GG, respectively. In most cases, the apparent persistence length, as determined by viscometry, indicated that aggregates remained in galactomannans after centrifugation and filtration. Results suggest an effective extraction time for each plant source of galactomannan based on its performance and its macromolecular behavior in solution.
Assuntos
Fabaceae/química , Mananas/isolamento & purificação , Sementes/química , Filtração , Galactose/análise , Mananas/química , Manose/análise , Peso Molecular , Espalhamento de Radiação , ViscosidadeRESUMO
We have investigated the interaction between the native neutral ß-cyclodextrin (CD) and the DNA molecule by performing single-molecule stretching experiments with optical tweezers. In particular, we have monitored the changes of the mechanical properties of the CD-DNA complexes as a function of the CD concentration in the sample. By using a quenched disorder statistical model, we were also capable to extract important physicochemical information (equilibrium binding constants, cooperativity degree) of such interaction from the mechanical data. In addition, we have found that the interaction occurs by two different mechanisms, first with the formation of relatively large CD clusters along the double helix, which thereafter can locally denature the DNA molecule by forming hydrogen bonds with the base pairs that eventually flip out. A prediction of our quenched disorder model was that cooperativity could be controlled by adjusting the surface charge of ß-CD molecules. This prediction is confirmed in the present work.