Photoreceptor Compartment-Specific TULP1 Interactomes

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, sev-eral OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach was to physically iso-late Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two dis-tinct interactomes. We report the identification of: 1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), 2) a synaptic-specific interaction be-tween Tulp1 and the scaffold protein Ribeye, and 3) an interaction between Tulp1 and the cyto-skeletal protein Microtubule-associated protein 1B (MAP1B) in both compartments. Immuno-localization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

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Photoreceptor Compartment-Specific TULP1 Interactomes

International Journal of Molecular Sciences ◽

10.3390/ijms22158066 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8066

Author(s):

Lindsey A. Ebke ◽

Satyabrata Sinha ◽

Gayle J. T. Pauer ◽

Stephanie A. Hagstrom

Keyword(s):

Molecular Motors ◽

Protein Trafficking ◽

Specific Interaction ◽

Specific Protein ◽

Vesicle Recycling ◽

Binding Partners ◽

Liquid Chromatography Tandem Mass ◽

In The Wild ◽

Specific Proteins ◽

Kinesin Family

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

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Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia

Human Molecular Genetics ◽

10.1093/hmg/ddab034 ◽

2021 ◽

Author(s):

Yamato Ishida ◽

Takuya Kobayashi ◽

Shuhei Chiba ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

Protein Trafficking ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Missense Variant ◽

Cellular Level ◽

Compound Heterozygous ◽

Hypomorphic Allele ◽

Genes Encoding ◽

Specific Proteins ◽

Compound Heterozygous Mutations

Abstract Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140, and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

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Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2011.2062 ◽

2011 ◽

Vol 279 (1733) ◽

pp. 1560-1566 ◽

Cited By ~ 71

Author(s):

Lee Koren ◽

Shinichi Nakagawa ◽

Terry Burke ◽

Kiran K. Soma ◽

Katherine E. Wynne-Edwards ◽

...

Keyword(s):

Stress Hormones ◽

Passer Domesticus ◽

House Sparrows ◽

Long Term Study ◽

Darwinian Fitness ◽

Liquid Chromatography Tandem Mass ◽

In The Wild ◽

The Subject ◽

As Stress

Potential mechanistic mediators of Darwinian fitness, such as stress hormones or sex hormones, have been the focus of many studies. An inverse relationship between fitness and stress or sex hormone concentrations has been widely assumed, although empirical evidence is scarce. Feathers gradually accumulate hormones during their growth and provide a novel way to measure hormone concentrations integrated over time. Using liquid chromatography–tandem mass spectrometry, we measured testosterone, corticosterone and cortisol in the feathers of house sparrows ( Passer domesticus ) in a wild population which is the subject of a long-term study. Although corticosterone is considered the dominant avian glucocorticoid, we unambiguously identified cortisol in feathers. In addition, we found that feathers grown during the post-nuptial moult in autumn contained testosterone, corticosterone and cortisol levels that were significantly higher in birds that subsequently died over the following winter than in birds that survived. Thus, feather steroids are candidate prospective biomarkers to predict the future survival of individuals in the wild.

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Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

The Journal of Cell Biology ◽

10.1083/jcb.81.1.123 ◽

1979 ◽

Vol 81 (1) ◽

pp. 123-136 ◽

Cited By ~ 18

Author(s):

N Agabian ◽

M Evinger ◽

G Parker

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Messenger Rna ◽

Cell Types ◽

Specific Protein ◽

Soluble Proteins ◽

Specific Cell ◽

Daughter Cells ◽

Specific Proteins ◽

Entire Cell

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.

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The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

10.1101/2020.12.21.423858 ◽

2020 ◽

Author(s):

Amrita Khakurel ◽

Tetyana Kudlyk ◽

Juan S. Bonifacino ◽

Vladimir V. Lupashin

Keyword(s):

Steady State ◽

Golgi Apparatus ◽

Protein Trafficking ◽

Essential Role ◽

Human Cell Lines ◽

Critical Component ◽

Intracellular Protein ◽

Vesicle Recycling ◽

The Stability ◽

Garp Complex

AbstractThe Golgi apparatus is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and vesicle recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits is detrimental to N- and O-glycosylation, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit impaired retention of glycosylation enzymes in the Golgi. Indeed, a RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi but instead is missorted to the endolysosomal compartment. We propose that the endosomal compartment is part of the trafficking itinerary of Golgi enzymes and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery.

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Modular protein-oligonucleotide signal exchange

Nucleic Acids Research ◽

10.1093/nar/gkaa405 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6431-6444

Author(s):

Deepak K Agrawal ◽

Rebecca Schulman

Keyword(s):

Specific Protein ◽

Vascular Endothelial ◽

Complementary Dna ◽

Exchange Processes ◽

Dna Strand Displacement ◽

Specific Proteins ◽

Dna Strand ◽

Endothelial Growth Factor ◽

Signal Exchange

Abstract While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.

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Unconventional Myosins: How Regulation Meets Function

International Journal of Molecular Sciences ◽

10.3390/ijms21010067 ◽

2019 ◽

Vol 21 (1) ◽

pp. 67 ◽

Cited By ~ 9

Author(s):

Natalia Fili ◽

Christopher P. Toseland

Keyword(s):

Molecular Motors ◽

Current Knowledge ◽

Local Environment ◽

Structural Features ◽

Regulatory Mechanisms ◽

Tight Control ◽

Cellular Processes ◽

Binding Partners ◽

Wide Range ◽

Multiple Levels

Unconventional myosins are multi-potent molecular motors that are assigned important roles in fundamental cellular processes. Depending on their mechano-enzymatic properties and structural features, myosins fulfil their roles by acting as cargo transporters along the actin cytoskeleton, molecular anchors or tension sensors. In order to perform such a wide range of roles and modes of action, myosins need to be under tight regulation in time and space. This is achieved at multiple levels through diverse regulatory mechanisms: the alternative splicing of various isoforms, the interaction with their binding partners, their phosphorylation, their applied load and the composition of their local environment, such as ions and lipids. This review summarizes our current knowledge of how unconventional myosins are regulated, how these regulatory mechanisms can adapt to the specific features of a myosin and how they can converge with each other in order to ensure the required tight control of their function.

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Plasma concentrations of platelet-specific proteins correlated with platelet survival

Blood ◽

10.1182/blood.v55.1.82.82 ◽

1980 ◽

Vol 55 (1) ◽

pp. 82-84 ◽

Cited By ~ 60

Author(s):

DJ Doyle ◽

CN Chesterman ◽

JF Cade ◽

JR McGready ◽

GC Rennie ◽

...

Keyword(s):

Life Span ◽

Plasma Concentrations ◽

Specific Protein ◽

Platelet Factor ◽

In Vivo Release ◽

Platelet Survival ◽

Specific Proteins ◽

Artery Disease ◽

Multiple Hit

Abstract Relationships between 51Cr platelet survival and plasma concentrations of beta-thromboglobulin (betaTG) and platelet factor 4 (PF4) were analyzed in 91 studies of patients with coronary artery disease. betaTG was significantly correlated with platelet life-span, turnover, and the number of hits in the multiple hit model. PF4 was significantly correlated with life-span and turnover. The most significant relationship involving platelet-specific protein concentrations and life-span estimates was between betaTG and life-span estimated using the multiple hit model (r = -0.39, p less than 0.001). There was a high correlation between betaTG and PF4 (r = 0.62, p less than 0.001), and no improvement could be obtained by combining the measurements of the two proteins in any regression with life-span or turnover. The results indicate that the patients with the shortest platelet survival time in this group tended to have the highest plasma concentration of betaTG and PF4 and thus probably increased in vivo release of betaTG and PF4. They strengthen the claim that these platelet-specific proteins may be indicators of platelet involvement in disease.

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Multiple mechanisms of dissociated epidermal cell spreading.

The Journal of Cell Biology ◽

10.1083/jcb.96.1.63 ◽

1983 ◽

Vol 96 (1) ◽

pp. 63-67 ◽

Cited By ~ 60

Author(s):

K S Stenn ◽

J A Madri ◽

T Tinghitella ◽

V P Terranova

Keyword(s):

Serum Albumin ◽

Epidermal Cell ◽

Type Iv Collagen ◽

Cell Spreading ◽

Specific Protein ◽

Epidermal Cells ◽

Type Iv ◽

Basement Membrane Protein ◽

Specific Proteins

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

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The STEP61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900878116 ◽

2019 ◽

Vol 116 (16) ◽

pp. 8028-8037 ◽

Cited By ~ 2

Author(s):

Sehoon Won ◽

Salvatore Incontro ◽

Yan Li ◽

Roger A. Nicoll ◽

Katherine W. Roche

Keyword(s):

Ampa Receptors ◽

Protein Tyrosine Phosphatase ◽

Protein Phosphatase ◽

Tyrosine Phosphatase ◽

Hippocampal Slices ◽

Specific Protein ◽

Synaptic Proteins ◽

Synaptic Currents ◽

Synaptic Regulation ◽

Binding Partners

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein phosphatase that regulates a variety of synaptic proteins, including NMDA receptors (NAMDRs). To better understand STEP’s effect on other receptors, we used mass spectrometry to identify the STEP61 interactome. We identified a number of known interactors, but also ones including the GluA2 subunit of AMPA receptors (AMPARs). We show that STEP61 binds to the C termini of GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. The synaptic expression of GluA2 and GluA3 is increased in STEP-KO mouse brain, and STEP knockdown in hippocampal slices increases AMPAR-mediated synaptic currents. Interestingly, STEP61 overexpression reduces the synaptic expression and synaptic currents of both AMPARs and NMDARs. Furthermore, STEP61 regulation of synaptic AMPARs is mediated by lysosomal degradation. Thus, we report a comprehensive list of STEP61 binding partners, including AMPARs, and reveal a central role for STEP61 in differentially organizing synaptic AMPARs and NMDARs.

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