CCSer2 activates dynein via Ndel1 inhibition. (A) Schematic depicting Ndel1 release from dynein, resulting in downstream formation of dynein transport complex. (B) Schematic of a binding assay to determine whether CCSer2^650–850 affects the dynein–Ndel1 interaction. (C) Schematic of the assay to determine whether CCSer2^650–850 affects Lis1-Ndel1 binding. (D) Percentage of dynein bound to Ndel1-conjugated beads in the absence (white circle) or presence (black circle) of CCSer2^650–850. n = 6. Error bars are the mean ± SD. Statistical analysis was performed with a Mann–Whitney test. (E) Percentage of Lis1 bound to Ndel1-conjugated beads in the absence (white circle) or presence (black circle) of CCSer2^650–850. n = 3. Error bars are the mean ± SD. Statistical analysis was performed with a Mann–Whitney test. (F) Median dissociation time of dynein–Ndel1 complexes in the absence (white circle) and presence (black circle) of 2 μM CCSer2^650–850. n = 225 for the condition without CCSer2^650–850 and 172 for the condition with CCSer2^650–850. These data were collected from seven separate experimental replicates. Error bars are 95% confidence intervals. Significance was determined from a Mann–Whitney test. (G) Quantification of DHC co-immunoprecipitation with FLAG-Ndel1 using α-FLAG resin out of U2OS WT or CCSer2-KO cells. n = 6 biological replicates. Error bars are the mean ± SEM, and the statistical analysis was determined with a ratio-paired t test. (H) Representative western blot of FLAG-Ndel1 co-immunoprecipitation experiments quantified in G, blotting for α-DHC, α-FLAG, and α-GAPDH. Input loaded is 1% of total cell lysate. (I) Model for CCSer2 delivery to the cortex and spatial regulation of dynein. (Step 1) CCSer2 binds to EB1 to associate with growing microtubule plus-ends to reach the cell periphery. (Step 2) Dynein–Lis1–Ndel1–dynactin form a primed structure that is poised for activation. It is also possible that dynein–Lis1–Ndel1 form a precomplex and dynactin associates after Ndel1 release. We have shown this pre-activation, primed structure on the microtubule, but it is also possible that this structure does not form with dynein bound directly to the microtubule. (Step 3) Ndel1-CCSer2 interaction reduces Ndel1’s affinity for dynein. (Step 4) Ndel1 release of dynein allows Lis1-mediated activation of dynein and dynactin association with dynein intermediate chain. (Step 5) The fully activated transport complex of dynein–dynactin–adaptor forms with adaptors that are already associated with cellular structures where active dynein will be recruited. (Cargo #1) Dynein–dynactin–adaptor complexes form on the actin cortex (adaptor unknown) to reposition the centrosome during migration. (Cargo #2) Dynein–dynactin–adaptor complexes form on early endosomes (likely Hook1 and Hook3) to drive retrograde trafficking. Interaction with adaptors drives dynein localization and activation. (J) Diagram illustrating the proposed tiered regulation of dynein activity. DHC, dynein heavy chain. Source data are available for this figure: SourceData F6.