scholarly journals RanGTP regulates cilium formation and ciliary trafficking of a kinesin-II subunit independent of its nuclear functions

2019 ◽  
Author(s):  
Shengping Huang ◽  
Prachee Avasthi
Keyword(s):  
Mammalian Cells ◽  
Intracellular Transport ◽  
Protein Targeting ◽  
Nuclear Translocation ◽  
Unicellular Eukaryote ◽  
Precise Control ◽  
Cilium Formation ◽  
Ciliary Protein ◽  
Cilia Formation ◽  
Protein Incorporation

AbstractKinesin is part of the microtubule (MT)-binding motor protein superfamily, which exerts crucial functions in cell division and intracellular transport in different organelles. The heterotrimeric kinesin-II, consisting of the kinesin like protein KIF3A/3B heterodimer and kinesin-associated protein KAP3, is highly conserved across species between the green alga Chlamydomonas and humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilium. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-II itself is poorly understood. Using the unicellular eukaryote Chlamydomonas and mammalian cells, we show that RanGTP regulates ciliary trafficking of KAP3. We found the armadillo repeat region 6-9 (ARM6-9) of KAP3, required for its nuclear translocation, is sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilia formation and the emerging roles of RanGTP/importin β in ciliary protein targeting, we further investigate the effect of RanGTP in cilium length regulation in these two different systems. We demonstrate that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to segregate RanGTP regulation of ciliary protein incorporation from of its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been coopted for independent roles in ciliary import.

scholarly journals Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit

2020 ◽  
pp. jbc.RA119.010936
Author(s):  
Shengping Huang ◽  
Larissa L Dougherty ◽  
Prachee Avasthi
Keyword(s):  
Protein Targeting ◽  
Nuclear Translocation ◽  
Nuclear Transport ◽  
Retinal Pigment Epithelial Cell ◽  
Retinal Pigment ◽  
Small Gtpase ◽  
Unicellular Eukaryote ◽  
Epithelial Cell Line ◽  
Cilium Formation ◽  
Ciliary Protein

Kinesin is part of the microtubule (MT)-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits KIF3A/3B and kinesin-associated protein KAP3, is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 itself are poorly understood. It has been previously suggested that ciliary transport is analogous to nuclear transport mechanisms.Using Chlamydomonas and hTERT-immortalized retinal pigment epithelial cell line, we show that RanGTP, a small GTPase that dictates nuclear transport, regulates ciliary trafficking of KAP3, a key component for functional kinesin-2. We found that the armadillo repeat region 6-9 (ARM6-9) of KAP3, required for its nuclear translocation, is also necessary and sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilium formation and there are the emerging roles for RanGTP/importin β in ciliary protein targeting, we further investigated the effect of RanGTP in cilium formation and maintenance. We found that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to provide orthogonal support in an algal model system that segregates RanGTP regulation of ciliary protein trafficking from its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been co-opted for independent roles in ciliary import.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

scholarly journals Cell-specific Activation of Nuclear Factor-κB by the ParasiteTrypanosoma cruziPromotes Resistance to Intracellular Infection

2000 ◽  
Vol 11 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Belinda S. Hall ◽  
Winnie Tam ◽  
Ranjan Sen ◽  
Miercio E. A. Pereira
Keyword(s):  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Microbial Pathogens ◽  
Tissue Tropism ◽  
Nuclear Factor ◽  
Infection Level ◽  
Direct Role ◽  
Intracellular Infection

The transcription factor nuclear factor-κB (NF-κB) is central to the innate and acquired immune response to microbial pathogens, coordinating cellular responses to the presence of infection. Here we demonstrate a direct role for NF-κB activation in controlling intracellular infection in nonimmune cells. Trypanosoma cruzi is an intracellular parasite of mammalian cells with a marked preference for infection of myocytes. The molecular basis for this tissue tropism is unknown. Trypomastigotes, the infectious stage of T. cruzi, activate nuclear translocation and DNA binding of NF-κB p65 subunit and NF-κB-dependent gene expression in epithelial cells, endothelial cells, and fibroblasts. Inactivation of epithelial cell NF-κB signaling by inducible expression of the inhibitory mutant IκBaM significantly enhances parasite invasion.T. cruzi do not activate NF-κB in cells derived from skeletal, smooth, or cardiac muscle, despite the ability of these cells to respond to tumor necrosis factor-α with NF-κB activation. The in vitro infection level in these muscle-derived cells is more than double that seen in the other cell types tested. Therefore, the ability of T. cruzi to activate NF-κB correlates inversely with susceptibility to infection, suggesting that NF-κB activation is a determinant of the intracellular survival and tissue tropism ofT. cruzi.

scholarly journals Nuclear Translocation of Soybean MPK6, GmMPK6, Is Mediated by Hydrogen Peroxide in Salt Stress

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2611
Author(s):  
Jong Hee Im ◽  
Seungmin Son ◽  
Jae-Heung Ko ◽  
Kyung-Hwan Kim ◽  
Chung Sun An ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Salt Stress ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Signaling Molecules ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction System ◽  
Diphenylene Iodonium ◽  
Mitogen Activated Protein

The plant mitogen-activated protein kinase (MPK) cascade, a highly conserved signal transduction system in eukaryotes, plays a crucial role in the plant’s response to environmental stimuli and phytohormones. It is well-known that nuclear translocation of MPKs is necessary for their activities in mammalian cells. However, the mechanism underlying nuclear translocation of plant MPKs is not well elucidated. In the previous study, it has been shown that soybean MPK6 (GmMPK6) is activated by phosphatidic acid (PA) and hydrogen peroxide (H2O2), which are two signaling molecules generated during salt stress. Using the two signaling molecules, we investigated how salt stress triggers its translocation to the nucleus. Our results show that the translocation of GmMPK6 to the nucleus is mediated by H2O2, but not by PA. Furthermore, the translocation was interrupted by diphenylene iodonium (DPI) (an inhibitor of RBOH), confirming that H2O2 is the signaling molecule for the nuclear translocation of GmMPK6 during salt stress.

Abstract 227: The Effect of PHLPP2 Removal on Cardiomyocyte Hypertrophy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Nicole H Purcell ◽  
Courtney Moc ◽  
Giovanni Birrueta ◽  
Amy Taylor ◽  
Walter Koch ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Preliminary Data ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Ph Domain ◽  
Akt Phosphorylation ◽  
Precise Control ◽  
Neonatal Cardiomyocytes ◽  
G Protein Coupled

Crucial cellular decisions that lead to cell growth, metabolism, proliferation, and survival are all dependent on the precise control of the phosphorylation state of proteins. The serine/threonine phosphatase, PHLPP (PH domain leucine-rich repeat protein phosphatase) has been shown to directly dephosphorylate several members of the AGC family of kinases. Knockdown of PHLPP1 by siRNA in neonatal cardiomyocytes potentiates Akt activity and phosphorylation specifically at Ser473 basally and following agonist stimulation while, the removal of PHLPP2 in cardiomyocytes does not affect Akt phosphorylation as previously reported in other cells. We hypothesize that PHLPP2 may target other AGC kinases in cardiomyocytes to regulate cardiac hypertrophy. Preliminary data suggests that removal of PHLPP2 activates fetal gene re-expression at baseline and potentiates phenylephrine (PE) induced gene expression 2 fold over siControl. Recently, G protein-coupled receptor kinase 5 (GRK5), which is an AGC kinase, has been shown to regulate cardiac hypertrophy through HDAC5 phosphorylation and de-repression of gene transcription. We wanted to determine whether PHLPP2 regulates GRK5 phosphorylation and localization in cardiomyocytes. GRK5 translocates to the nucleus following hypertrophic stimulation and we found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and with PE treatment compared to siControl cells. Also, removal of PHLPP2 increased nuclear export of HDAC5 at baseline and following PE treatment. Conversely, overexpression of PHLPP2 blocked nuclear translocation of GRK5 following PE treatment. Ongoing studies will determine whether PHLPP acts as a scaffold or if its phosphatase activity is necessary for inhibition of GRK5 translocation by directly measuring the phosphorylation of GRK5 in the presence and absence of PHLPP2 following hypertrophic stimulation. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non-canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

2001 ◽  
Vol 114 (10) ◽  
pp. 1893-1900 ◽  
Author(s):  
S. Lusa ◽  
T.S. Blom ◽  
E.L. Eskelinen ◽  
E. Kuismanen ◽  
J.E. Mansson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Ldl Cholesterol ◽  
Normal Cells ◽  
Recycling Endosomes ◽  
Regulate Protein ◽  
Niemann Pick ◽  
Accumulation Characteristic

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

scholarly journals Functional domains of interferon regulatory factor I (IRF-1)

1998 ◽  
Vol 335 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Fred SCHAPER ◽  
Sabine KIRCHHOFF ◽  
Guido POSERN ◽  
Mario KÖSTER ◽  
André OUMARD ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Functional Domains ◽  
Dna Binding Domain ◽  
Transcriptional Activation Domain

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitroand was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

scholarly journals Light-Mediated Control over TRPC3-Mediated NFAT Signaling

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 556 ◽  
Author(s):  
Annarita Graziani ◽  
Bernadett Bacsa ◽  
Denis Krivic ◽  
Patrick Wiedner ◽  
Sanja Curcic ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Nuclear Translocation ◽  
Receptor Potential ◽  
Hek293 Cells ◽  
Trpc Channels ◽  
Activated T Cells ◽  
Precise Control ◽  
All Optical ◽  
Tight Linkage ◽  
Transient Receptor

Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

scholarly journals Induction of Ran GTP drives ciliogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4539-4548 ◽  
Author(s):  
Shuling Fan ◽  
Eileen L. Whiteman ◽  
Toby W. Hurd ◽  
Jeremy C. McIntyre ◽  
John F. Dishinger ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Basal Bodies ◽  
Ciliary Protein ◽  
Cilia Formation ◽  
Ran Gtp ◽  
New Evidence ◽  
Gtpase Ran

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin–Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.

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