Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

Morris, Robert L.; English, Christopher N.; Lou, Julia; Dufort, Fay J.; Nordberg, Joshua J.; Terasaki, Mark; Hinkle, Beth

doi:10.1016/j.ydbio.2004.06.017

Cited by 26 publications

(24 citation statements)

References 79 publications

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“…Confocal microscopy of living cells confirmed that KAP-GFP-labeled particles can be observed to move in both directions along the length of the flagella, similar to that reported previously for KAP-GFP in the sensory cilia of the nematode, C. elegans (Signor et al, 1999). KAP-GFP signals have also been observed in blastula cilia of sea urchin embryos (Morris et al, 2004). Interestingly, the Chlamydomonas KAP-GFP subunit is more efficiently incorporated into the Kinesin-2 complex of the flagella than the fla3 mutant subunit, even though both are present at similar levels in the cell body.…”

Section: Rescue Of Fla3 Defects With Epitope-tagged Kap Sequencesmentioning

confidence: 97%

TheFLA3KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

Mueller

Perrone

Bower

et al. 2005

MBoC

133

150

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Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella. INTRODUCTIONCilia and flagella perform essential motile and sensory functions for a variety of eukaryotic organisms. In vertebrates, defects in ciliary and flagellar motility (primary ciliary dyskinesia or PCD) result in randomization of left/right body asymmetry during embryonic development, chronic respiratory disease, and male sterility (Ibañ ez-Tallon et al., 2003). Defects in the assembly of primary cilia or cilia-associated proteins have also been linked to polycystic kidney disease (PKD), retinal degeneration, hearing loss, and human obesity disorders . Studies in Chlamydomonas, Caenorhabditis elegans, and mice have demonstrated that the machinery required for ciliary assembly is highly conserved (Rosenbaum and Witman, 2002). In most cases, the assembly and maintenance of these organelles depends on a bidirectional, intraflagellar transport (IFT) system of large protein particles driven by two distinct microtubule motors, a heterotrimeric Kinesin-2 and a novel cytoplasmic dynein (cDhc1b or Dhc2; Cole, 2003). Formerly known as kinesin-II (Lawrence et al., 2004), Kinesin-2 is responsible for transport from the basal body region to the tip of the axoneme (anterograde IFT; Kozminski et al., 1995;Piperno and Mead, 1997;Cole et al., 1998), whereas the cDhc1b/LIC complex is required for transport from the flagellar tip to the basal body region (retrograde IFT; Pazour et al., 1999;Porter et al., 1999;Signor et al., 1999a;Wicks et al., 2000;Perrone et al., 2003;Schafer et al., 2003).The Kinesin-2 complex consists of two distinct kinesinrelated motor subunits and a third kinesin-associated protein (KAP). This complex was first described in sea urchin eggs and subsequently identified in the mouse, Chlamydomonas, C. elegans, Dros...

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Section: Rescue Of Fla3 Defects With Epitope-tagged Kap Sequencesmentioning

confidence: 97%

TheFLA3KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

Mueller

Perrone

Bower

et al. 2005

MBoC

133

150

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“…The first fluorescent protein fusion movement of IFT was described by Orozco et al (1999) in the nematode Caenorhabditis elegans demonstrating the processive motion of IFT particles in living sensory neuronal cilia. Since that time a number of systems have been used to observe IFT imaging via fluorescent protein fusions including Chlamydomonas (Mueller et al , 2005), trypanosomes (Absalon et al , 2008), sea urchin (Morris et al , 2004), and cultured mammalian cell lines (Follit et al , 2006; Nachury et al , 2007; Tran et al , 2008). Central to these observations has been the ability to dissect particle velocities as well as distinctions between anterograde and retrograde motility.…”

Section: Rationalementioning

confidence: 99%

Imaging Intraflagellar Transport in Mammalian Primary Cilia

Besschetnova¹,

Roy²,

Shah³

2009

Methods in Cell Biology

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The primary cilium is a specialized organelle that projects from the surface of many cell types. Unlike its motile counterpart it cannot beat but does transduce extracellular stimuli into intracellular signals and acts as a specialized subcellular compartment. The cilium is built and maintained by the transport of proteins and other biomolecules into and out of this compartment. The trafficking machinery for the cilium is referred to as IFT or intraflagellar transport. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella. Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations. Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling.

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“…Several NLS-like sequences have been found on the KIF3A/KIF3B/KAP complex, and the KAP subunit has been observed to redistribute from cilia nuclei during the mitotic cycle40. It is likely that additional signals in KIF17 are required to promote ciliary rather than nuclear import.…”

mentioning

confidence: 99%

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-β2 and RanGTP

et al. 2010

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The biogenesis, maintenance, and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor1,2. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. We identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLS) suggests that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a Ran-GTP gradient across the ciliary base. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with importin-β2 in a manner dependent on the CLS and inhibited by Ran-GTP. We propose that Ran plays a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments.

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Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

Cited by 26 publications

References 79 publications

TheFLA3KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

TheFLA3KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

Imaging Intraflagellar Transport in Mammalian Primary Cilia

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-β2 and RanGTP

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