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LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.

Functional interplay between LIS1, NDE1 and NDEL1 in dynein-dependent organelle positioning