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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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Coexpression of EB1 increases the pool of extended, active KIF17 in MDCK cells. (A) Fluorescence images of mCh-KIF17-EmGFP expressed alone or with Myc-EB1. Red mask shows the localization of active mCh-KIF17-EmGFP. Bar, 20 µm. (B and D) Box–whisker plots showing the distribution of FRETeff under each experimental condition. (C and E) Box–whisker plots showing the fraction of active KIF17 under each experimental condition. 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.
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fig3: Coexpression of EB1 increases the pool of extended, active KIF17 in MDCK cells. (A) Fluorescence images of mCh-KIF17-EmGFP expressed alone or with Myc-EB1. Red mask shows the localization of active mCh-KIF17-EmGFP. Bar, 20 µm. (B and D) Box–whisker plots showing the distribution of FRETeff under each experimental condition. (C and E) Box–whisker plots showing the fraction of active KIF17 under each experimental condition. 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.

Mentions: We showed previously that localization of endogenous KIF17 to MT plus ends in epithelial cells, where it could participate in cortical MT stabilization, is dependent on EB1 but that mutant KIF17G754E accumulates at plus ends and stabilizes MTs independent of EB1 (Jaulin and Kreitzer, 2010). This led us to speculate that EB1 binding to KIF17 at MT ends could either activate the motor for MT stabilization or enhance its accumulation in an active form at MT ends. To test this directly, we coexpressed mCh-KIF17-EmGFP and Myc-EB1 in MDCK cells and analyzed KIF17 conformation with FLIM (Fig. 3). In these experiments, EB1 coexpression resulted in a 20% decrease in EAV as compared with control cells expressing KIF17 alone (Fig. 3 B and Table 1). Phasor analysis showed this change in FRET efficiency corresponded to a 55% increase in the population of extended KIF17 when EB1 was coexpressed (Fig. 3 C and Table 1).


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)

Coexpression of EB1 increases the pool of extended, active KIF17 in MDCK cells. (A) Fluorescence images of mCh-KIF17-EmGFP expressed alone or with Myc-EB1. Red mask shows the localization of active mCh-KIF17-EmGFP. Bar, 20 µm. (B and D) Box–whisker plots showing the distribution of FRETeff under each experimental condition. (C and E) Box–whisker plots showing the fraction of active KIF17 under each experimental condition. 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824023&req=5

fig3: Coexpression of EB1 increases the pool of extended, active KIF17 in MDCK cells. (A) Fluorescence images of mCh-KIF17-EmGFP expressed alone or with Myc-EB1. Red mask shows the localization of active mCh-KIF17-EmGFP. Bar, 20 µm. (B and D) Box–whisker plots showing the distribution of FRETeff under each experimental condition. (C and E) Box–whisker plots showing the fraction of active KIF17 under each experimental condition. 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.
Mentions: We showed previously that localization of endogenous KIF17 to MT plus ends in epithelial cells, where it could participate in cortical MT stabilization, is dependent on EB1 but that mutant KIF17G754E accumulates at plus ends and stabilizes MTs independent of EB1 (Jaulin and Kreitzer, 2010). This led us to speculate that EB1 binding to KIF17 at MT ends could either activate the motor for MT stabilization or enhance its accumulation in an active form at MT ends. To test this directly, we coexpressed mCh-KIF17-EmGFP and Myc-EB1 in MDCK cells and analyzed KIF17 conformation with FLIM (Fig. 3). In these experiments, EB1 coexpression resulted in a 20% decrease in EAV as compared with control cells expressing KIF17 alone (Fig. 3 B and Table 1). Phasor analysis showed this change in FRET efficiency corresponded to a 55% increase in the population of extended KIF17 when EB1 was coexpressed (Fig. 3 C and Table 1).

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