Probing the structure of cytoplasm.

Luby-Phelps, Katherine; Taylor, D. Lansing; Lanni, Frederick

doi:10.1083/jcb.102.6.2015

Cited by 321 publications

(267 citation statements)

References 45 publications

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“…The diffusion coefficients of FITC-dextran were intermediate between the two aldolase values and in good agreement with published in vivo values for similarly sized dextrans (44). The lack of difference in the perinuclear and peripheral mobilities of FITC-dextran rules out significant effects of cell geometry on measured values of D~y~o.…”

Section: Spatial Differences In Aldolase Mobility Exist In Vivosupporting

confidence: 87%

“…FRAP experiments were performed by the Gaussian spot method of Axelrod et al (9) essentially as described previously (43,44). The 514 tun (for rhodamine) and 488 nm (for fluorescein) lines of an ion argon laser (Spectra Physics, Inc.) were focused through a Universal microscope equipped with epifluorescence optics (Carl Zeiss, Inc., Thornwood, NY).…”

Section: Frap and Calculations Of Diffusion Coefficientsmentioning

confidence: 99%

“…Bleaching times ranged from 5 to 50 ms; 15-25% bleaching gave the most consistent data. Spot radii, measured as described previously (43,44), were between 6 and 22 ~tm, and fluorescence recovery was monitored for 12-18 s, which represented 15-20 half-times of recovery. FRAP curves were analyzed by the method of Yguerabide et al (81) or Van Zoelen et al (73); the two methods agreed well when equally good curve fits were compared.…”

Section: Frap and Calculations Of Diffusion Coefficientsmentioning

confidence: 99%

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Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Pagliaro

Taylor

1988

The Journal of Cell Biology

115

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Abstract. We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 × 10 -7 cm2/s, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 × 10 -7 cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 × 10 -g cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITCdextran indicated that FITC-dextran was relatively excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.TIaOUGH glycolytic metabolism is well-defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been characterized to date. After the elucidation of mitochondrial structure and function 35 years ago (28) it was generally assumed that a corresponding organelle for glycolysis must exist. Efforts to isolate and to characterize such an organelle were unsuccessful, leading de Duve (26) to conclude that the longsought"glycolytic particle" did not exist. As a result, the current concept of in vivo glycolytic metabolism is based on an assumption of dilute solution chemistry, due largely to the absence of evidence for a discrete glycolytic organelle.There has been evidence for over 20 years that some glycoiytic enzymes bind to structural proteins, particularly actin.

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Section: Spatial Differences In Aldolase Mobility Exist In Vivosupporting

confidence: 87%

Section: Frap and Calculations Of Diffusion Coefficientsmentioning

confidence: 99%

Section: Frap and Calculations Of Diffusion Coefficientsmentioning

confidence: 99%

See 1 more Smart Citation

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Pagliaro

Taylor

1988

The Journal of Cell Biology

115

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“…The diffusion coefficients for rapid secretory vesicles in growth cones are a thousandfold smaller than expected for diffusion in water, but are similar to those found for inert, similar-sized beads in cytoplasm (K. Luby-Phelps, personal communication). Thus, it is possible that the fast vesicles we detected are moving by Brownian motion and that their speed is limited by the meshwork of cytoskeleton and the viscosity of the cytoplasm (20). If this is the case, it would suggest that mechanisms for directing movement may be reserved for supporting fast release from small, synaptic vesicles at neuronal sites of release.…”

Section: Discussionmentioning

confidence: 98%

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Han

Axelrod

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

124

154

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Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

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“…In a living cell, the cytoplasm is filled with structures of different sizes such that diffusion is limited and actually dependent on the size of the diffusing object [231,295]. Furthermore, vesicles in axonal transport usually originate at the synapse or in the cell body, so that conservation of transported particles is provided within the axon.…”

Section: Finite Diffusion: a Two-species Tasep Coupled To A Diffusivementioning

confidence: 99%

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

2015

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Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized.The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach.It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer.We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the transportation of large cargos by multiple motors, we concentrate on axonal transport, because of its relevance for neuronal diseases. Some important characteristics of axonal transport is that it takes place in a confined environment; Besides several types of motors are involved, that move in opposite directions. It is a challenge to understand how this bidirectional transport is organized. We review several features that could contribute to the efficiency of bidirectional transport in the axon, including in particular the role of motor-motor interactions and of the dynamics of the underlying microtubule network. Finally, we also discuss some open questions that may be relevant for future research in this field.

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Probing the structure of cytoplasm.

Cited by 321 publications

References 45 publications

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

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