Quantitative imaging of Rac1 activity in Dictyostelium cells with a fluorescently labelled GTPase-binding domain from DPAKa kinase

ABSTRACTTo build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin, and both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends requires a C-terminal region outside NuMA’s canonical microtubule binding domain, and it is independent of direct minus-end binders γ-TuRC, CAMSAP1, and KANSL1/3. Both NuMA’s minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

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NuMA recruits dynein activity to microtubule minus-ends at mitosis

eLife ◽

10.7554/elife.29328 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 35

Author(s):

Christina L Hueschen ◽

Samuel J Kenny ◽

Ke Xu ◽

Sophie Dumont

Keyword(s):

Steady State ◽

Laser Ablation ◽

Quantitative Imaging ◽

Binding Domain ◽

Terminal Region ◽

Spindle Microtubule ◽

Microtubule Binding ◽

Bipolar Spindle ◽

Microtubule Binding Domain ◽

Spindle Microtubules

To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA’s canonical microtubule-binding domain and is independent of minus-end binders γ-TuRC, CAMSAP1, and KANSL1/3. Both NuMA’s minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

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EphA4 Signaling Regulates Blastomere Adhesion in the Xenopus Embryo by Recruiting Pak1 to Suppress Cdc42 Function

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0294 ◽

2007 ◽

Vol 18 (3) ◽

pp. 1030-1043 ◽

Cited By ~ 19

Author(s):

Nicolas Bisson ◽

Luc Poitras ◽

Alexander Mikryukov ◽

Michel Tremblay ◽

Tom Moss

Keyword(s):

Catalytic Activity ◽

Cell Adhesion ◽

Cell Sorting ◽

Ectopic Expression ◽

Binding Domain ◽

Dominant Negative ◽

Membrane Targeting ◽

Dependent Manner ◽

Eph Receptors ◽

Gtpase Binding Domain

The control of cell adhesion is an important mechanism by which Eph receptors regulate cell sorting during development. Activation of EphA4 in Xenopus blastulae induces a reversible, cell autonomous loss-of-adhesion and disruption of the blastocoel roof. We show this phenotype is rescued by Nckβ (Grb4) dependent on its interaction with EphA4. Xenopus p21Cdc42/Rac-activated kinase xPAK1 interacts with Nck, is activated in embryo by EphA4 in an Nck-dependent manner, and is required for EphA4-induced loss-of-adhesion. Ectopic expression of xPAK1 phenocopies EphA4 activation. This does not require the catalytic activity of xPAK1, but it does require its GTPase binding domain and is enhanced by membrane targeting. Indeed, membrane targeting of the GTPase binding domain (GBD) of xPAK1 alone is sufficient to phenocopy EphA4 loss-of-adhesion. Both EphA4 and the xPAK1-GBD down-regulate RhoA-GTP levels, and consistent with this, loss-of-adhesion can be rescued by activated Cdc42, Rac, and RhoA and can be epistatically induced by dominant-negative RhoA. Despite this, neither Cdc42 nor Rac activities are down-regulated by EphA4 activation or by the xPAK1-GBD. Together, the data suggest that EphA4 activation sequesters active Cdc42 and in this way down-regulates cell–cell adhesion. This novel signaling pathway suggests a mechanism for EphA4-guided migration.

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