Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains

Janson, L W; Ragsdale, Keith; Luby-Phelps, Katherine

doi:10.1016/s0006-3495(96)79367-0

Cited by 44 publications

(41 citation statements)

References 28 publications

(32 reference statements)

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“…Protein concentrations may be regulated by protein-protein association or by changes in compartment morphology, as has been demonstrated in, for example, dendritic spines. Whereas the potential implications of steric volume exclusion have been recognized (10)(11), its impact on cell signaling has been inaccessible to direct study due primarily to the fact that most of these compartments are smaller than can be resolved with current live cell fluorescence imaging technologies. The light-controlled modulation of arrestin levels in the ciliary rod outer segment, combined with the fact that this signaling compartment is large enough to allow quantitative fluorescence imaging, make photoreceptors an unparalleled model system for studying these mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Najafi¹,

Maza²,

Calvert

2011

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

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Proteins segregate into discrete subcellular compartments via a variety of mechanisms, including motor protein transport, local binding, and diffusion barriers. This physical separation of cell functions serves, in part, as a mechanism for controlling compartment activity by allowing regulation of local protein concentrations. In this study we explored how soluble protein size impacts access to the confined space within the retinal photoreceptor outer segment signaling compartment and discovered a strikingly steep relationship. We find that GFP monomers, dimers, and trimers expressed transgenically in frog rods are present in the outer segment at 1.8-, 2.9-, and 6.8-fold lower abundances, relative to the cell body, than the small soluble fluorescent marker, calcein. Theoretical analysis, based on statistical-mechanical models of molecular access to polymer meshes, shows that these observations can be explained by the steric hindrance of molecules occupying the highly constrained spaces between outer segment disc membranes. This mechanism may answer a long-standing question of how the soluble regulatory protein, arrestin, is effectively excluded from the outer segments of dark-adapted rods and cones. Generally, our results suggest an alternate mode for the control of protein access to cell domains based on dynamic, sizedependent compartmental partitioning that does not require diffusion barriers, active transport, or large numbers of immobile binding sites.any cellular functions take place in highly spatially constrained compartments such as primary cilia, microvilli, endoplasmic reticulum (ER)/Golgi, filopodia, or dendritic spines. Delivery and retention of membrane-associated proteins to these areas are mediated by several complementary mechanisms, including intraflagellar transport (1), local binding to relatively immobile structures (2), and barriers to diffusion between the specialized compartment and the rest of the cell (3-4). Although it is generally recognized that the activities of these compartments are regulated by soluble proteins, especially in the case of cell signaling (5), specific rules governing soluble protein access to the compartments are not well defined. We addressed this problem in rod photoreceptors of Xenopus laevis where visual signal transduction occurs within the narrow confines of a sensory cilium.Photoreceptor outer segments are ciliary organelles containing a stack of up to ∼2,000 flattened membrane vesicles called lamellar discs (5). The main protein component of these membranes is the visual pigment rhodopsin. Most other signaling proteins acting downstream of rhodopsin are tethered to the discs by posttranslational lipidation or single-pass transmembrane anchorage (6). An exception is arrestin, a soluble protein involved in the termination of the rhodopsin-mediated light response (7). An important feature of photoresponse regulation by arrestin is that arrestin levels in the outer segment are modulated in a light-dependent manner. Arrestin is nearly undetectable in the outer se...

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Section: Discussionmentioning

confidence: 99%

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Najafi¹,

Maza²,

Calvert

2011

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

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“…This rapid rate is similar to the rate at which the oligos move in aqueous solution and indicates that diffusion can occur in the nucleus at rates approximating those measured in solution. Although the viscosity of the cytoplasm has been investigated (24)(25)(26)(27), only a few measurements of molecular diffusion rates in the nucleus have been reported, primarily in isolated nuclei (20,28,29). Some of these studies report that nuclear viscosity is greater than water, and others suggest that molecular movement in the nucleus occurs at aqueous solution rates.…”

Section: Discussionmentioning

confidence: 99%

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Politz

Browne

Wolf

et al. 1998

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

253

209

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Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by f luorescence correlation spectroscopy (FCS) and f luorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 ؋ 10 ؊7 cm 2 ͞s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (<1 ؋ 10 ؊7 cm 2 ͞s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 ؋ 10 ؊7 cm 2 ͞s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.An understanding of the physical environment inside the cell nucleus is central to a coherent view of gene expression. It is important to know how the viscosity and molecular diffusion rates in the nucleus of a living cell compare with experimental conditions in vitro, where interactions between nucleic acids and proteins are studied at high dilution in aqueous solution. For example, it is not clear whether ribonucleoprotein (RNP) complexes can diffuse freely about the nucleus, impeded only by locally high concentrations of macromolecules or, alternatively, are bound or compartmentalized in such a way as to constrain their motion. Some observations suggest that premRNA transcripts are tethered to elements of the transcriptional, splicing, and͞or polyadenylation machinery (1-3), and it has been proposed that processed mRNAs make their way out of the nucleus by molecular diffusion (4, 5). However, mediated processes have not been ruled out, and the functional relationships between RNA export and nuclear structure remain unclear (6).Recently, Politz et al. (7) characterized nucleic acid uptake and hybridization in living cells by in situ reverse transcription and found that fluorescently labeled oligo(dT) can be taken up by living cells and form hybrid...

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“…The mechanisms that establish and maintain this polarity remain poorly defined, especially in the case of soluble proteins, but ultimately depend on a detailed understanding the cytoplasmic space through which macromolecules move. Efforts to characterize the cytoplasmic space with fluorescent molecular probes of various sizes have revealed a sizedependent partitioning into different subcellular regions (Janson et al, 1996;Luby-Phelps, 2000). The most likely explanation of this partitioning is 'sieving', i.e.…”

Section: Introductionmentioning

confidence: 99%

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

Peet

Bragin

Calvert

et al. 2004

Journal of Cell Science

101

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The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

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Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains

Cited by 44 publications

References 28 publications

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

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