Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

Calvert, Peter D.; Schiesser, William E.; Pugh, Edward N.

doi:10.1083/jcb1885oia11

Cited by 13 publications

(21 citation statements)

References 18 publications

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“…Essentially two mechanisms have been postulated: a diffusion barrier in the ciliary transition zone that would prevent arrestin from entering the outer segment without motor protein transport (22,23) and a binding partner, other than rhodopsin, that would localize arrestin to the inner segment (24). Our recent study, bolstered by results presented here, effectively ruled out the diffusion barrier hypothesis (25). Three binding partners have been identified for arrestin in dark-adapted rods, α-tubulin (24), enolase 1 (26), and NSF (27).…”

Section: Discussionsupporting

confidence: 55%

“…Animal experiments were approved by the State University of New York Upstate Committee for the Humane Use of Animals and carried out in accordance with the Guide for the Care and Use of Laboratory Animals (1996; National Academy of Sciences, Washington, DC). Quantitative imaging and multiphoton FRAPb and FRAPa experiments were conducted using a custom-built confocal and multiphoton microscope described previously (16,25). Detailed materials and methods are available in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 55%

Section: Methodsmentioning

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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“…1). The cilium does not appear to provide a significant barrier to diffusion (25), and proteins equilibrate across the cilium within 3 min (26); that is, much faster than the time scale of the measurements presented here. Although the reasons for the leveling off are not clear, factors that could possibly be playing a role include the finite size of the NADP pool, saturation of the metabolic enzymes, and perhaps cellular control of the [NADPH]/[NADP ϩ ] ratio.…”

Section: Discussionmentioning

confidence: 68%

Mitochondria Contribute to NADPH Generation in Mouse Rod Photoreceptors

Adler

Chen

Koutalos

2014

Journal of Biological Chemistry

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Background:The major source of NADPH is considered to be the pentose phosphate pathway. Results: In isolated mouse rod photoreceptors, blocking metabolite entry into mitochondria substantially reduces NADPH generation. Mitochondrial metabolic substrates support NADPH generation. Conclusion: Mitochondria-linked pathways contribute substantially to NADPH generation in mouse rod photoreceptors. Significance: A wide range of photoreceptor cell functions depend on adequate NADPH supply.

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“…The diffusion of soluble molecules between the ciliary lumen and the cytoplasm has only been examined in photoreceptor cells, and the 27 kDa green fluorescent protein (GFP, a barrel 4.2 nm long by 2.4 nm diameter) can equilibrate between frog photoreceptors’ inner and outer segments within ~3 min, very close to the theoretical value of 3.5 min assuming free diffusion through the connecting cilium (Nair et al 2005, Calvert et al 2006, Calvert et al 2010). Remarkably, it is now thought that the vectorial transport of arrestin, the rhodopsin desensitizer, and transducins Gα and Gβγ between the inner and outer segments are driven solely by diffusion through the connecting cilium and by retention at binding sites provided by rhodopsin in the outer segment (Calvert et al 2006).…”

Section: Is the Cilium A Cellular Organelle?mentioning

confidence: 86%

Trafficking to the Ciliary Membrane: How to Get Across the Periciliary Diffusion Barrier?

Nachury

Seeley

Jin

2010

Annu. Rev. Cell Dev. Biol.

400

459

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The primary cilium organizes numerous signal transduction cascades and an understanding of signaling receptors trafficking to cilia is now emerging. A defining feature of cilia is the periciliary diffusion barrier that separates the ciliary and plasma membranes despite the topological continuity between these two membranes. Although lateral transport through this barrier may take place, polarized exocytosis to the base of the cilium has been the prevailing model for delivering membrane proteins to cilia. Key players for this polarized exocytosis model include the GTPases Rab8 and Rab11, the exocyst and possibly the intraflagellar tranport machinery. Sorting membrane proteins to cilia critically relies on the recognition of ciliary targeting signals by sorting machines such as the BBSome coat complex or the GTPase Arf4. Finally, signaling at the cilium entails the bidirectional movement of proteins between cytoplasm and cilia and ubiquitination may promote exit from cilia. Keywords CILIA; FLAGELLA; SIGNALING DIFFUSION BARRIERTHE CENTRAL PROBLEM: SEPARATION BETWEEN THE SIGNALING COMPARTMENT AND THE SITE OF PROTEIN SYNTHESISCilia and flagella are some of the most ancient eukaryotic structures and thus define the cenancestral eukaryote in the same way as nuclei and mitochondria do (Cavalier-Smith 2002). Although the structural organization of cilia and flagella appears fairly invariant, consisting of a core of nine microtubule doublets ensheathed within the ciliary membrane, the functions of cilia and flagella are remarkably diverse. Flagella and motile cilia (these Copyright © 2010 by Annual Reviews. All rights reserved DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. FUTURE ISSUESWhere is the sorting decision made for proteins targeted to the cilium versus the plasma membrane? Are ciliary membrane proteins packaged into vesicles distinct from those of plasma membrane resident proteins at the level of the TGN, or is there an intermediate step (plasma membrane, recycling endosomes) in sorting? What is the molecular machinery that recognizes ciliary targeting sequences and sorts proteins to the cilium? How is this machinery regulated? This and the previous question are likely to shed light on the mechanisms that regulate the dynamic trafficking of signaling proteins (e.g., Smo) to the primary cilium. What is the molecular nature of the periciliary diffusion barrier? It will be important to determine how many membrane proteins reach the cilium by crossing this barrier versus utilizing the polarized exocytosis route. How are proteins removed from cilia? Is there a specific machinery involved in endocytosis of ciliary proteins? Is this step regulated by ubiquitination? NIH Public Access Author ManuscriptAnnu Rev Cell Dev Biol. Author manuscript; available in PMC 2011 November 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript terms can be used interchange...

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Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

Cited by 13 publications

References 18 publications

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Mitochondria Contribute to NADPH Generation in Mouse Rod Photoreceptors

Trafficking to the Ciliary Membrane: How to Get Across the Periciliary Diffusion Barrier?

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