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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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Localization of active and inactive populations of KIF17 in MDCK cells. (A) Immunostaining of tyrosinated and detyrosinated tubulin in cells microinjected with mCh-KIF17-EmGFP (outlined cells and inset) and treated for 45 min with 33 µM NZ. (B) Representative FLIM phasor analysis of mCh-KIF17-EmGFP in cells. Fluorescence image (inset) and the distribution of KIF17 in extended, active (red mask on image and red circle on phasor plot, FRETeff < 7%) and compact, inactive (green mask on image and green circle on phasor plot, FRETeff = 7–21%) forms determined from measured lifetimes. Inset on the phasor plot shows analysis of mCh-KIF17G754E-EmGFP. (C) Representative FLIM phasor analysis of mCh-KIF17G754E-EmGFP in cells as in B. (D) Box–whisker plots showing the distribution of FRETeff and populations of active and inactive mCh-KIF17-EmGFP and mCh-KIF17G754E-EmGFP in in MDCK cells. Data represent the indicated number of cells (n) obtained from three or more independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (E) Quantification of immunoblots probed for detyrosinated (Glu) and acetylated (Ace) tubulin in control or mCh-KIF17-EmGFP transfected cells. Ratio of tubulin/actin was normalized to 1 ± SD in controls. Data are derived from five experiments. Bars, 20 µm.
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fig1: Localization of active and inactive populations of KIF17 in MDCK cells. (A) Immunostaining of tyrosinated and detyrosinated tubulin in cells microinjected with mCh-KIF17-EmGFP (outlined cells and inset) and treated for 45 min with 33 µM NZ. (B) Representative FLIM phasor analysis of mCh-KIF17-EmGFP in cells. Fluorescence image (inset) and the distribution of KIF17 in extended, active (red mask on image and red circle on phasor plot, FRETeff < 7%) and compact, inactive (green mask on image and green circle on phasor plot, FRETeff = 7–21%) forms determined from measured lifetimes. Inset on the phasor plot shows analysis of mCh-KIF17G754E-EmGFP. (C) Representative FLIM phasor analysis of mCh-KIF17G754E-EmGFP in cells as in B. (D) Box–whisker plots showing the distribution of FRETeff and populations of active and inactive mCh-KIF17-EmGFP and mCh-KIF17G754E-EmGFP in in MDCK cells. Data represent the indicated number of cells (n) obtained from three or more independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (E) Quantification of immunoblots probed for detyrosinated (Glu) and acetylated (Ace) tubulin in control or mCh-KIF17-EmGFP transfected cells. Ratio of tubulin/actin was normalized to 1 ± SD in controls. Data are derived from five experiments. Bars, 20 µm.

Mentions: We first determined that N- and C-terminal tags did not interfere with KIF17 function by testing whether FRET constructs stabilized MTs when expressed in MDCK cells, as described previously for the constitutively active, extended conformation hinge mutant GFP-KIF17G754E (Jaulin and Kreitzer, 2010). In cells expressing mCh-KIF17-EmGFP, KIF17-EmGFP, GFP-KIF17, or mCh-KIF17G754E-EmGFP, we observed an increase in nocodazole (NZ)-resistant, posttranslationally modified (detyrosinated or acetylated) stable MTs (Fig. 1, A and E; and not depicted). Thus, fluorescently tagged wild-type and mutant KIF17 are active for MT stabilization, demonstrating that these constructs are functional in 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)

Localization of active and inactive populations of KIF17 in MDCK cells. (A) Immunostaining of tyrosinated and detyrosinated tubulin in cells microinjected with mCh-KIF17-EmGFP (outlined cells and inset) and treated for 45 min with 33 µM NZ. (B) Representative FLIM phasor analysis of mCh-KIF17-EmGFP in cells. Fluorescence image (inset) and the distribution of KIF17 in extended, active (red mask on image and red circle on phasor plot, FRETeff < 7%) and compact, inactive (green mask on image and green circle on phasor plot, FRETeff = 7–21%) forms determined from measured lifetimes. Inset on the phasor plot shows analysis of mCh-KIF17G754E-EmGFP. (C) Representative FLIM phasor analysis of mCh-KIF17G754E-EmGFP in cells as in B. (D) Box–whisker plots showing the distribution of FRETeff and populations of active and inactive mCh-KIF17-EmGFP and mCh-KIF17G754E-EmGFP in in MDCK cells. Data represent the indicated number of cells (n) obtained from three or more independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (E) Quantification of immunoblots probed for detyrosinated (Glu) and acetylated (Ace) tubulin in control or mCh-KIF17-EmGFP transfected cells. Ratio of tubulin/actin was normalized to 1 ± SD in controls. Data are derived from five experiments. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Localization of active and inactive populations of KIF17 in MDCK cells. (A) Immunostaining of tyrosinated and detyrosinated tubulin in cells microinjected with mCh-KIF17-EmGFP (outlined cells and inset) and treated for 45 min with 33 µM NZ. (B) Representative FLIM phasor analysis of mCh-KIF17-EmGFP in cells. Fluorescence image (inset) and the distribution of KIF17 in extended, active (red mask on image and red circle on phasor plot, FRETeff < 7%) and compact, inactive (green mask on image and green circle on phasor plot, FRETeff = 7–21%) forms determined from measured lifetimes. Inset on the phasor plot shows analysis of mCh-KIF17G754E-EmGFP. (C) Representative FLIM phasor analysis of mCh-KIF17G754E-EmGFP in cells as in B. (D) Box–whisker plots showing the distribution of FRETeff and populations of active and inactive mCh-KIF17-EmGFP and mCh-KIF17G754E-EmGFP in in MDCK cells. Data represent the indicated number of cells (n) obtained from three or more independent experiments ± SEM. Box–whisker plots show minimum, 25th percentile, median, 75th percentile, maximum, and mean FRET values. (E) Quantification of immunoblots probed for detyrosinated (Glu) and acetylated (Ace) tubulin in control or mCh-KIF17-EmGFP transfected cells. Ratio of tubulin/actin was normalized to 1 ± SD in controls. Data are derived from five experiments. Bars, 20 µm.
Mentions: We first determined that N- and C-terminal tags did not interfere with KIF17 function by testing whether FRET constructs stabilized MTs when expressed in MDCK cells, as described previously for the constitutively active, extended conformation hinge mutant GFP-KIF17G754E (Jaulin and Kreitzer, 2010). In cells expressing mCh-KIF17-EmGFP, KIF17-EmGFP, GFP-KIF17, or mCh-KIF17G754E-EmGFP, we observed an increase in nocodazole (NZ)-resistant, posttranslationally modified (detyrosinated or acetylated) stable MTs (Fig. 1, A and E; and not depicted). Thus, fluorescently tagged wild-type and mutant KIF17 are active for MT stabilization, demonstrating that these constructs are functional in 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