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A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation.

Espenel C, Acharya BR, Kreitzer G - J. Cell Biol. (2013)

Bottom Line: Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells.Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia.Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Weill Cornell Medical College of Cornell University, New York, NY 10021.

ABSTRACT
We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy transfer. Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells. Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia. Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

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PKC activity is required for KIF17 activation in MDCK cells. (A) Box–whisker plots showing the distribution of FRETeff (left) and the fraction of active KIF17 (right) in control and 20 µM BIM-1–treated cells (1 h). Data were obtained from at least three independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (B) Immunostaining of detyrosinated and tyrosinated tubulin in cells expressing mCh-KIF17-EmGFP after cDNA injection (outlined cells and inset) and treatment with BIM-1 as in A. Bars, 10 µm. (C) GFP-KIF17G754E localizes at the plus ends of MTs in cell protrusions (arrows) in control and BIM-1–treated cells as in B. Cells were fixed 2 h after cDNA microinjection. Inj, injection. Bars, 20 µm.
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fig2: PKC activity is required for KIF17 activation in MDCK cells. (A) Box–whisker plots showing the distribution of FRETeff (left) and the fraction of active KIF17 (right) in control and 20 µM BIM-1–treated cells (1 h). Data were obtained from at least three independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (B) Immunostaining of detyrosinated and tyrosinated tubulin in cells expressing mCh-KIF17-EmGFP after cDNA injection (outlined cells and inset) and treatment with BIM-1 as in A. Bars, 10 µm. (C) GFP-KIF17G754E localizes at the plus ends of MTs in cell protrusions (arrows) in control and BIM-1–treated cells as in B. Cells were fixed 2 h after cDNA microinjection. Inj, injection. Bars, 20 µm.

Mentions: PKC family members have been implicated in regulation of MT dynamics and polarization of several cell types and act in the cortical MT capture and stabilization pathway (Kabir et al., 2001; Fan et al., 2004; Ruiz-Canada et al., 2004; Eng et al., 2006). Because KIF17 may act through this pathway (Jaulin and Kreitzer, 2010), we tested whether PKC affects KIF17 conformation and activity for MT stabilization. We treated MDCK cells expressing mCh-KIF17-EmGFP with the PKC inhibitor BIM-1 (bisindolylmaleimide I; 20 µM) for 1 h (Fig. 2); at this concentration, classical PKCs α and βI and novel PKCs δ and ε are likely inhibited (Martiny-Baron et al., 1993).


A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation.

Espenel C, Acharya BR, Kreitzer G - J. Cell Biol. (2013)

PKC activity is required for KIF17 activation in MDCK cells. (A) Box–whisker plots showing the distribution of FRETeff (left) and the fraction of active KIF17 (right) in control and 20 µM BIM-1–treated cells (1 h). Data were obtained from at least three independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (B) Immunostaining of detyrosinated and tyrosinated tubulin in cells expressing mCh-KIF17-EmGFP after cDNA injection (outlined cells and inset) and treatment with BIM-1 as in A. Bars, 10 µm. (C) GFP-KIF17G754E localizes at the plus ends of MTs in cell protrusions (arrows) in control and BIM-1–treated cells as in B. Cells were fixed 2 h after cDNA microinjection. Inj, injection. Bars, 20 µm.
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fig2: PKC activity is required for KIF17 activation in MDCK cells. (A) Box–whisker plots showing the distribution of FRETeff (left) and the fraction of active KIF17 (right) in control and 20 µM BIM-1–treated cells (1 h). Data were obtained from at least three independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (B) Immunostaining of detyrosinated and tyrosinated tubulin in cells expressing mCh-KIF17-EmGFP after cDNA injection (outlined cells and inset) and treatment with BIM-1 as in A. Bars, 10 µm. (C) GFP-KIF17G754E localizes at the plus ends of MTs in cell protrusions (arrows) in control and BIM-1–treated cells as in B. Cells were fixed 2 h after cDNA microinjection. Inj, injection. Bars, 20 µm.
Mentions: PKC family members have been implicated in regulation of MT dynamics and polarization of several cell types and act in the cortical MT capture and stabilization pathway (Kabir et al., 2001; Fan et al., 2004; Ruiz-Canada et al., 2004; Eng et al., 2006). Because KIF17 may act through this pathway (Jaulin and Kreitzer, 2010), we tested whether PKC affects KIF17 conformation and activity for MT stabilization. We treated MDCK cells expressing mCh-KIF17-EmGFP with the PKC inhibitor BIM-1 (bisindolylmaleimide I; 20 µM) for 1 h (Fig. 2); at this concentration, classical PKCs α and βI and novel PKCs δ and ε are likely inhibited (Martiny-Baron et al., 1993).

Bottom Line: Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells.Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia.Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Weill Cornell Medical College of Cornell University, New York, NY 10021.

ABSTRACT
We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy transfer. Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells. Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia. Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.

Show MeSH
Related in: MedlinePlus