scholarly journals Transient accumulation and bidirectional movement of KIF13B in primary cilia

2021 ◽  
Author(s):  
Alice Dupont Juhl ◽  
Zeinab Anvarian ◽  
Julia Berges ◽  
Daniel Wustner ◽  
Lotte B Pedersen
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Primary Cilia ◽  
Cell Imaging ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Ciliary Membrane ◽  
Bidirectional Movement ◽  
And Function ◽  
Male Specific

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

scholarly journals Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

2018 ◽  
Author(s):  
Martin F. Engelke ◽  
Bridget Waas ◽  
Sarah E. Kearns ◽  
Ayana Suber ◽  
Allison Boss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Genetic Approach ◽  
Ciliated Cells ◽  
Loss Of Function ◽  
Double Knockout ◽  
Chemical Genetic ◽  
Species Specific ◽  
And Function

ABSTRACTThe trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemical-genetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition. These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function.

scholarly journals Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at the base of primary cilia and suppresses ciliary elongation

2021 ◽  
Author(s):  
Stine K Morthorst ◽  
Camilla Nielsen ◽  
Pietro Farinelli ◽  
Zeinab Anvarian ◽  
Christina B. R. Rasmussen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Primary Cilia ◽  
Cell Imaging ◽  
Vesicle Transport ◽  
Imaging Analysis ◽  
Ciliary Function ◽  
Cargo Transport ◽  
Direct Binding ◽  
Crumbs Complex

The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to KIF13Bs MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live cell imaging analysis indicated that Ap80 is concentrated at the base of primary cilia and recruits PALS1 to this site, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia and altered IGF-1 signaling in cultured mammalian cells. Our results suggest that Ap80 may specifically activate KIF13B cargo binding at the base of primary cilia to regulate ciliary length and signaling.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

2015 ◽  
Vol 166 (1-4) ◽  
pp. 101-103 ◽  
Author(s):  
M. Noguchi ◽  
Y. Kanari ◽  
A. Yokoya ◽  
A. Narita ◽  
K. Fujii
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Study ◽  
Live Cell ◽  
Mitochondrial Morphology ◽  
X Rays

scholarly journals MyTH4-FERM myosins have an ancient and conserved role in filopod formation

2016 ◽  
Vol 113 (50) ◽  
pp. E8059-E8068 ◽  
Author(s):  
Karl J. Petersen ◽  
Holly V. Goodson ◽  
Ashley L. Arthur ◽  
G. W. Gant Luxton ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Null Mutant ◽  
Minimal Elements ◽  
Social Amoeba ◽  
Functional Conservation ◽  
Functional Homolog ◽  
The Social ◽  
Functional Features

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.

scholarly journals Live cell imaging reveals differential modifications to cytoplasmic dynein properties by phospho- and dephosphomimic mutations of the intermediate chain 2C S84

2014 ◽  
Vol 92 (9) ◽  
pp. 1143-1154 ◽  
Author(s):  
Kiev R. Blasier ◽  
Michael K. Humsi ◽  
Junghoon Ha ◽  
Mitchell W. Ross ◽  
W. Russell Smiley ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cytoplasmic Dynein ◽  
Live Cell ◽  
Intermediate Chain

Intermediate chain subunit as a probe for cytoplasmic dynein function: Biochemical analyses and live cell imaging in PC12 cells

2007 ◽  
Vol 85 (12) ◽  
pp. 2640-2647 ◽  
Author(s):  
Kenneth R. Myers ◽  
Kevin W.-H. Lo ◽  
R. John Lye ◽  
John M. Kogoy ◽  
Violetta Soura ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cytoplasmic Dynein ◽  
Live Cell ◽  
Biochemical Analyses ◽  
Intermediate Chain

scholarly journals DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xinxin Wang ◽  
Zhiming Chen ◽  
Marcel Mettlen ◽  
Jungsik Noh ◽  
Sandra L Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Single Channel ◽  
Accessory Proteins ◽  
Sensitive Detector ◽  
Coated Pits ◽  
Misleading Information ◽  
Bona Fide ◽  
Biochemical Measurements

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.

scholarly journals Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

2017 ◽  
Author(s):  
Noa Aloush ◽  
Tomer Schvartz ◽  
Andres I. König ◽  
Sarit Cohen ◽  
Eugene Brozgol ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Stop Codon ◽  
Signal To Noise Ratio ◽  
Trna Synthetase ◽  
Live Cell ◽  
Live Cells ◽  
Copy Numbers ◽  
Intracellular Proteins

ABSTRACTGenetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/ pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

scholarly journals Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

2019 ◽  
Author(s):  
Darren Machin ◽  
Daniel Williamson ◽  
Peter Fisher ◽  
victoria miller ◽  
Gemma Wildsmith ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Neuronal Tracing ◽  
Future Role ◽  
Golgi Localization

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase-labelling approach to generate site-specifically <i>N</i>-terminally modified variants of both the A2-B<sub>5</sub> heterohexamer and B<sub>5</sub> pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B<sub>5</sub> pentamer showed an unexpected localization in the <i>medial/trans</i> Golgi. This study suggests a future role for specifically-labelled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labelling of lipid-rafts in fixed cells.

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