Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of ...microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.
Display omitted
▸ Laser ablation reveals microtubule length and organization in the spindle ▸ Microtubule lengths are inhomogeneous and shortest near poles ▸ The stability of microtubules is not spatially regulated ▸ Spindle microtubules are organized by spatially varying nucleation and transport
A new laser-ablation method reveals the organization of microtubules within the Xenopus spindle and suggests a model for spindle organization involving spatially regulated nucleation and transport of microtubules.
The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly ...coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell’s metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling.
Display omitted
•PI3K signaling positively regulates aldolase activity in epithelial cells•PI3K activation mobilizes aldolase from F-actin, increasing flux through glycolysis•PI3K-to-aldolase signaling occurs through Rac and not through AKT•PI3K coordinates cytoskeletal dynamics and glycolysis in vitro and in vivo
Phosphoinositide 3-kinase directly coordinates glycolysis by activating Rac, which remodels the actin cytoskeleton to free actin-bound aldolase.
The Physics of the Metaphase Spindle Oriola, David; Needleman, Daniel J; Brugués, Jan
Annual review of biophysics,
05/2018, Letnik:
47, Številka:
1
Journal Article
Recenzirano
The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other ...microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.
Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in ...the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.
Understanding the coordination of cell-division timing is one of the outstanding questions in the field of developmental biology. One active control parameter of the cell-cycle duration is ...temperature, as it can accelerate or decelerate the rate of biochemical reactions. However, controlled experiments at the cellular scale are challenging, due to the limited availability of biocompatible temperature sensors, as well as the lack of practical methods to systematically control local temperatures and cellular dynamics. Here, we demonstrate a method to probe and control the cell-division timing in Caenorhabditis elegans embryos using a combination of local laser heating and nanoscale thermometry. Local infrared laser illumination produces a temperature gradient across the embryo, which is precisely measured by in vivo nanoscale thermometry using quantum defects in nanodiamonds. These techniques enable selective, controlled acceleration of the cell divisions, even enabling an inversion of division order at the two-cell stage. Our data suggest that the cell-cycle timing asynchrony of the early embryonic development in C. elegans is determined independently by individual cells rather than via cell-to-cell communication. Our method can be used to control the development of multicellular organisms and to provide insights into the regulation of cell-division timings as a consequence of local perturbations.
Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, ...the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.
Cytoplasmic dynein-1 is a minus-end-directed motor protein that transports cargo over long distances and organizes the intracellular microtubule (MT) network. How dynein motor activity is harnessed ...for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, resulting in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, suggesting that minus-end accumulations of motors produce network-scale contractile stresses. Our data provide a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the cytoskeleton, for example, during mitotic spindle assembly.
Display omitted
•Dynein-dynactin cooperatively form limited-sized motor clusters at MT minus-ends•End clusters provide spatial restriction of force production to MT minus-ends•End clusters slide MTs without orientation bias, leading to minus-end focusing•End clusters produce contractile forces on millimeter-scale MT networks
Tan et al. demonstrate how individual cytoplasmic dynein motors organize incoherent collections of microtubules into polarity-sorted structures at varying length scales. Dynein cooperatively accumulates into clusters at microtubule minus-ends, reorganizing microtubules exclusively via clusters, thus providing a molecular explanation for dynein's structural role in mitotic spindle assembly.
Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of ...cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.
Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, ...and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the fluorescence lifetime imaging microscopy measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand of the cell do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of a spatially heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.
PDF
