scholarly journals Sequences in the stalk domain regulate autoinhibition and ciliary tip localization of the immotile kinesin-4 KIF7

2021 ◽  
Author(s):  
T. Lynne Blasius ◽  
Yang Yue ◽  
Raghu Ram Prasad ◽  
Xinglei Liu ◽  
Arne Gennerich ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Human Disease ◽  
Hedgehog Signaling ◽  
Coiled Coil ◽  
Full Length ◽  
Microtubule Binding ◽  
Stalk Domain ◽  
In Cells

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for autoinhibition of KIF7 whereas the adjacent regulatory coiled coil (rCC) that contributes to autoinhibition of the motile kinesin-4's KIF21A and KIF21B, is not sufficient for KIF7 autoinhibition. Disease-associated mutations in the inhCC relieve autoinhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC and could be increased by disease-associated mutations regardless of the protein's autoinhibition state. These findings suggest that loss of KIF7 autoinhibition and/or altered cilium tip localization can contribute to pathogenesis of human disease.

scholarly journals Microtubule binding of the kinesin-4 KIF7 and its regulation by autoinhibition

2019 ◽  
Author(s):  
T. Lynne Blasius ◽  
Yang Yue ◽  
Kristen Verhey
Keyword(s):  
Growth Rates ◽  
Mammalian Cells ◽  
Hedgehog Signaling ◽  
Microtubule Dynamics ◽  
Microtubule Binding ◽  
Cell Extracts ◽  
Microtubule Growth ◽  
Linker Domain ◽  
In Cells

AbstractKIF7 is a member of the kinesin-4 family and plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length motor cannot bind to microtubules whereas truncated versions bind statically to microtubules in cells. Previous work suggested that truncated KIF7 motors bind preferentially to the plus ends of microtubules in vitro, however, we find that truncated KIF7 does not bind preferentially to or track the plus ends of growing microtubules in mammalian cells or in cell extracts. Although the truncated KIF7 did alter microtubule dynamics in cells, this property is not specific to KIF7 as expression of an active kinesin-1 motor also altered microtubule growth rates. The immotile behavior of KIF7 is not due to the extended neck linker domain as its deletion does not activate KIF7 for motility and its presence in a KIF5C/KIF7 chimeric motor does not prevent processive motility. Together this work indicates that the atypical kinesin KIF7 is regulated by autoinhibition to prevent binding to microtubules and alteration of microtubule dynamics in cells.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals Tripartite Motif 16 Inhibits the Migration and Invasion in Ovarian Cancer Cells

2017 ◽  
Vol 25 (4) ◽  
pp. 551-558 ◽  
Author(s):  
Hongwei Tan ◽  
Jin Qi ◽  
Guanghua Chu ◽  
Zhaoyang Liu
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Hedgehog Signaling ◽  
Epithelial Mesenchymal Transition ◽  
Coiled Coil ◽  
Migration And Invasion ◽  
Ovarian Cancer Cells ◽  
Mesenchymal Transition ◽  
Tripartite Motif

Tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite motif (TRIM) protein family, has been shown to play a role in tumor development and progression. However, the role of TRIM16 in ovarian cancer has never been revealed. Thus, in this study, we investigated the roles and mechanisms of TRIM16 in ovarian cancer. Our results demonstrated that TRIM16 expression was low in ovarian cancer cell lines. In addition, overexpression of TRIM16 significantly inhibited the migration and invasion in vitro, as well as suppressed the epithelial‐mesenchymal transition (EMT) phenotype in ovarian cancer cells. Furthermore, overexpression of TRIM16 greatly inhibited the protein expression levels of Shh, Smo, Ptc, Gli-1, MMP2, and MMP9 in ovarian cancer cells. Taken together, these results strongly suggest that TRIM16 inhibits the migration and invasion via suppressing the Sonic hedgehog signaling pathway in ovarian cancer cells. Thus, TRIM16 may be a novel potential therapeutic target for ovarian cancer.

Hedgehog-induced ciliary trafficking of kinesin-4 motor KIF7 requires intraflagellar transport but not KIF7’s microtubule binding

2021 ◽  
Author(s):  
Yang Yue ◽  
Martin F. Engelke ◽  
T. Lynne Blasius ◽  
Kristen J. Verhey
Keyword(s):  
Transcription Factors ◽  
Signaling Pathway ◽  
Primary Cilium ◽  
Hedgehog Signaling ◽  
Intraflagellar Transport ◽  
Hedgehog Signaling Pathway ◽  
Microtubule Binding ◽  
Pathway Activation ◽  
Gli Transcription Factors ◽  
Ciliary Localization

The kinesin-4 motor KIF7 is a conserved regulator of the Hedgehog signaling pathway. In vertebrates, Hedgehog signaling requires the primary cilium, and KIF7 and Gli transcription factors accumulate at the cilium tip in response to Hedgehog activation. Unlike conventional kinesins, KIF7 is an immotile kinesin and its mechanism of ciliary accumulation is unknown. We generated KIF7 variants with altered microtubule binding or motility. We demonstrate that microtubule binding of KIF7 is not required for the increase in KIF7 or Gli localization at the cilium tip in response to Hedgehog signaling. In addition, we show that the immotile behavior of KIF7 is required to prevent ciliary localization of Gli transcription factors in the absence of Hedgehog signaling. Using an engineered kinesin-2 motor that enables acute inhibition of intraflagellar transport (IFT), we demonstrate that kinesin-2 KIF3A/KIF3B/KAP mediates the translocation of KIF7 to the cilium tip in response to Hedgehog pathway activation. Together, these results suggest that KIF7’s role at the tip of the cilium is unrelated to its ability to bind to microtubules.

scholarly journals Intracellular cargo transport by single-headed kinesin motors

2019 ◽  
Vol 116 (13) ◽  
pp. 6152-6161 ◽  
Author(s):  
Kristin I. Schimert ◽  
Breane G. Budaitis ◽  
Dana N. Reinemann ◽  
Matthew J. Lang ◽  
Kristen J. Verhey
Keyword(s):  
Intracellular Transport ◽  
Motor Coordination ◽  
Optical Trap ◽  
Coiled Coil ◽  
Force Output ◽  
Cellular Assays ◽  
Cellular Events ◽  
Cargo Transport ◽  
In Cells

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

scholarly journals MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

2020 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Atsushi Suzuki
Keyword(s):  
Coiled Coil ◽  
Golgi Membrane ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Ribbon Structure ◽  
Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

scholarly journals Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

1999 ◽  
Vol 147 (7) ◽  
pp. 1569-1582 ◽  
Author(s):  
Michelangelo Cordenonsi ◽  
Fabio D'Atri ◽  
Eva Hammar ◽  
David A.D. Parry ◽  
John Kendrick-Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Coiled Coil ◽  
Full Length ◽  
Cytoplasmic Surface ◽  
Terminal Fragment ◽  
Reticulocyte Lysates ◽  
Actomyosin Cytoskeleton

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

scholarly journals Coronin Promotes the Rapid Assembly and Cross-linking of Actin Filaments and May Link the Actin and Microtubule Cytoskeletons in Yeast

1999 ◽  
Vol 144 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Bruce L. Goode ◽  
Jonathan J. Wong ◽  
Anne-Christine Butty ◽  
Matthias Peter ◽  
Ashley L. McCormack ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Coiled Coil ◽  
Cross Linking ◽  
Cortical Actin ◽  
Functional Link ◽  
Microtubule Binding ◽  
Filament Assembly ◽  
Cell Extracts ◽  
Cross Links

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 × 10−9 M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Δ deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Δ cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

scholarly journals Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

2005 ◽  
Vol 25 (9) ◽  
pp. 3715-3725 ◽  
Author(s):  
Jan Liman ◽  
Sundar Ganesan ◽  
Christoph P. Dohm ◽  
Stan Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Heat Shock Protein 70 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Full Length ◽  
Wild Type ◽  
Binding Characteristics ◽  
In Cells

ABSTRACT It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGΔC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGΔC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGΔC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGΔC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

scholarly journals The actin nucleation factor JMY is a negative regulator of neuritogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4563-4574 ◽  
Author(s):  
Elif Nur Firat-Karalar ◽  
Peter P. Hsiue ◽  
Matthew D. Welch
Keyword(s):  
Cell Lines ◽  
Regulatory Protein ◽  
Neuronal Cell ◽  
Full Length ◽  
Negative Regulator ◽  
Neurite Formation ◽  
A Minor ◽  
And Function ◽  
In Cells

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.

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