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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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Active KIF17 is on stable MTs in MDCK cells. Box–whisker plots showing the distribution of KIF17 FRETeff (left) and the fraction of active KIF17 (right) 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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fig4: Active KIF17 is on stable MTs in MDCK cells. Box–whisker plots showing the distribution of KIF17 FRETeff (left) and the fraction of active KIF17 (right) 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: The constitutively active, extended mutant GFP-KIF17G754E accumulates in large puncta at MT plus ends near the cell cortex in epithelial cells. This localization is observed even in cells treated with NZ to depolymerize dynamic MTs (Jaulin and Kreitzer, 2010), suggesting that active KIF17 is on stable MTs. To determine whether extended KIF17 localizes on a specific subset of MTs, we analyzed mCh-KIF17-EmGFP by FLIM when only dynamic MTs or both dynamic and stable MTs were depolymerized. Depolymerization of dynamic MTs had no effect on EAV or on the fraction of extended conformation mCh-KIF17-EmGFP as compared with untreated controls (Fig. 4, A and B). However, when both dynamic and stable MTs were depolymerized, we measured a 30% increase in EAV accompanied by a 43% decrease in the extended population of KIF17 when compared with control untreated cells (Fig. 4, A and B). To break down stable, modified MTs selectively, we treated cells expressing mCh-KIF17-EmGFP with 100 nM okadaic acid (OA) for 90 min (Fig. S2 A; Gurland and Gundersen, 1993). OA induced a 42% decrease in the extended population of KIF17 to 15.9 ± 2.6% (Fig. S2, B and C), similar to what we observed in cells treated with cold and NZ. Although OA depolymerizes stable, modified MTs preferentially in MDCK cells, we cannot yet exclude the possibility that it affects KIF17 conformation and activity by modifying phosphorylation of the kinesin directly or of another factor that modulates KIF17 activity in our cells.


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)

Active KIF17 is on stable MTs in MDCK cells. Box–whisker plots showing the distribution of KIF17 FRETeff (left) and the fraction of active KIF17 (right) 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

fig4: Active KIF17 is on stable MTs in MDCK cells. Box–whisker plots showing the distribution of KIF17 FRETeff (left) and the fraction of active KIF17 (right) 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: The constitutively active, extended mutant GFP-KIF17G754E accumulates in large puncta at MT plus ends near the cell cortex in epithelial cells. This localization is observed even in cells treated with NZ to depolymerize dynamic MTs (Jaulin and Kreitzer, 2010), suggesting that active KIF17 is on stable MTs. To determine whether extended KIF17 localizes on a specific subset of MTs, we analyzed mCh-KIF17-EmGFP by FLIM when only dynamic MTs or both dynamic and stable MTs were depolymerized. Depolymerization of dynamic MTs had no effect on EAV or on the fraction of extended conformation mCh-KIF17-EmGFP as compared with untreated controls (Fig. 4, A and B). However, when both dynamic and stable MTs were depolymerized, we measured a 30% increase in EAV accompanied by a 43% decrease in the extended population of KIF17 when compared with control untreated cells (Fig. 4, A and B). To break down stable, modified MTs selectively, we treated cells expressing mCh-KIF17-EmGFP with 100 nM okadaic acid (OA) for 90 min (Fig. S2 A; Gurland and Gundersen, 1993). OA induced a 42% decrease in the extended population of KIF17 to 15.9 ± 2.6% (Fig. S2, B and C), similar to what we observed in cells treated with cold and NZ. Although OA depolymerizes stable, modified MTs preferentially in MDCK cells, we cannot yet exclude the possibility that it affects KIF17 conformation and activity by modifying phosphorylation of the kinesin directly or of another factor that modulates KIF17 activity in our cells.

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