scholarly journals Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

2014 ◽  
Vol 207 (2) ◽  
pp. 269-282 ◽  
Author(s):  
Michael Taschner ◽  
Fruzsina Kotsis ◽  
Philipp Braeuer ◽  
E. Wolfgang Kuehn ◽  
Esben Lorentzen
Keyword(s):  
Crystal Structures ◽  
Molecular Motors ◽  
Mdck Cells ◽  
Intraflagellar Transport ◽  
Dominant Negative ◽  
Tetratricopeptide Repeat ◽  
Core Complex ◽  
Disease Protein ◽  
In Trans ◽  
And Function

Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281–329 in a complex that interacts directly with IFT70/IFT52330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.

scholarly journals Redundant and Distinct Functions for Dynamin-1 and Dynamin-2 Isoforms

1998 ◽  
Vol 143 (7) ◽  
pp. 1871-1881 ◽  
Author(s):  
Yoram Altschuler ◽  
Shana M. Barbas ◽  
Laura J. Terlecky ◽  
Kitty Tang ◽  
Stephen Hardy ◽  
...  
Keyword(s):  
Hela Cells ◽  
Membrane Trafficking ◽  
Splice Variants ◽  
Mdck Cells ◽  
Dominant Negative ◽  
Vesicle Formation ◽  
Apical Surface ◽  
Receptor Mediated Endocytosis ◽  
And Function ◽  
Dynamin 2

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

scholarly journals Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery

2018 ◽  
Vol 62 (6) ◽  
pp. 753-763 ◽  
Author(s):  
Jenna L. Wingfield ◽  
Karl-Ferdinand Lechtreck ◽  
Esben Lorentzen
Keyword(s):  
Membrane Proteins ◽  
Molecular Motors ◽  
Intraflagellar Transport ◽  
Inherited Disease ◽  
Cell Organelles ◽  
Bardet Biedl Syndrome ◽  
Ciliary Membrane ◽  
Peripheral Membrane Proteins ◽  
Abnormal Accumulation ◽  
And Function

Bardet–Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.

scholarly journals ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mona Alsolami ◽  
Stefanie Kuhns ◽  
Manal Alsulami ◽  
Oliver E. Blacque
Keyword(s):  
Sonic Hedgehog ◽  
Primary Cilium ◽  
Molecular Motors ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Signaling Proteins ◽  
Sonic Hedgehog Signaling ◽  
And Function

Abstract Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

scholarly journals Intraflagellar transport molecules in ciliary and nonciliary cells of the retina

2010 ◽  
Vol 189 (1) ◽  
pp. 171-186 ◽  
Author(s):  
Tina Sedmak ◽  
Uwe Wolfrum
Keyword(s):  
Molecular Motors ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Retinal Neurons ◽  
Core Complex ◽  
Cytoplasmic Transport ◽  
Transport Vesicles ◽  
Transport Molecules ◽  
Established Role ◽  
Intense Research

The assembly and maintenance of cilia require intraflagellar transport (IFT), a process mediated by molecular motors and IFT particles. Although IFT is a focus of current intense research, the spatial distribution of individual IFT proteins remains elusive. In this study, we analyzed the subcellular localization of IFT proteins in retinal cells by high resolution immunofluorescence and immunoelectron microscopy. We report that IFT proteins are differentially localized in subcompartments of photoreceptor cilia and in defined periciliary target domains for cytoplasmic transport, where they are associated with transport vesicles. IFT20 is not in the IFT core complex in photoreceptor cilia but accompanies Golgi-based sorting and vesicle trafficking of ciliary cargo. Moreover, we identify a nonciliary IFT system containing a subset of IFT proteins in dendrites of retinal neurons. Collectively, we provide evidence to implicate the differential composition of IFT systems in cells with and without primary cilia, thereby supporting new functions for IFT beyond its well-established role in cilia.

Crystal Structures of Fragments D and Double-D from Fibrinogen and Fibrin

1999 ◽  
Vol 82 (08) ◽  
pp. 271-276 ◽  
Author(s):  
Glen Spraggon ◽  
Stephen Everse ◽  
Russell Doolittle
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Crystal Structures ◽  
Structure And Function ◽  
X Ray ◽  
Long Period ◽  
And Function

IntroductionAfter a long period of anticipation,1 the last two years have witnessed the first high-resolution x-ray structures of fragments from fibrinogen and fibrin.2-7 The results confirmed many aspects of fibrinogen structure and function that had previously been inferred from electron microscopy and biochemistry and revealed some unexpected features. Several matters have remained stubbornly unsettled, however, and much more work remains to be done. Here, we review several of the most significant findings that have accompanied the new x-ray structures and discuss some of the problems of the fibrinogen-fibrin conversion that remain unresolved. * Abbreviations: GPR—Gly-Pro-Arg-derivatives; GPRPam—Gly-Pro-Arg-Pro-amide; GHRPam—Gly-His-Arg-Pro-amide

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

scholarly journals Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

1998 ◽  
Vol 142 (1) ◽  
pp. 101-115 ◽  
Author(s):  
Tzuu-Shuh Jou ◽  
Eveline E. Schneeberger ◽  
W. James Nelson
Keyword(s):  
Tight Junctions ◽  
Spatial Organization ◽  
Mdck Cells ◽  
Protein Localization ◽  
Freeze Fracture ◽  
Tracer Diffusion ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Solute Diffusion ◽  
Fence Function

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.

scholarly journals CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance

2009 ◽  
Vol 206 (2) ◽  
pp. 421-434 ◽  
Author(s):  
Randall H. Friedline ◽  
David S. Brown ◽  
Hai Nguyen ◽  
Hardy Kornfeld ◽  
JinHee Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Cytotoxic T Lymphocyte ◽  
Critical Role ◽  
Lymphoproliferative Disease ◽  
In Trans ◽  
And Function

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.

scholarly journals A disease-associated frameshift mutation in caveolin-1 disrupts caveolae formation and function through introduction of a de novo ER retention signal

2017 ◽  
Vol 28 (22) ◽  
pp. 3095-3111 ◽  
Author(s):  
Courtney A. Copeland ◽  
Bing Han ◽  
Ajit Tiwari ◽  
Eric D. Austin ◽  
James E. Loyd ◽  
...  
Keyword(s):  
Frameshift Mutation ◽  
De Novo ◽  
Dominant Negative ◽  
Caveolin 1 ◽  
Protein Levels ◽  
C Terminus ◽  
Er Retention ◽  
And Function ◽  
Er Retention Signal ◽  
Retention Signal

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1–/– MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1–/– MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.

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