Fig 2: NDE1 or NDEL1 overexpression largely rescues the neuronal migration defects seen after knockdown of either protein.To test for RNAi rescue and functional complementation, embryonic rat brains were co-electroporated at E16 with shRNA to the various conditions with cDNA encoding RNAi-resistant proteins for self-rescue, and standard cDNA for cross-rescue. All the analyses were done at E20. Quantification of the amount of electroporated cells migrating into the cortical plate (CP) is shown at right. (a) Overexpression of NDE1 and NDEL1 produced no significant change in the amount of neurons migrating into the CP. (b) NDE1 knockdown with overexpression of RNAi-resistant NDE1 rescued neuronal migration into the CP. Overexpression of NDEL1 during NDE1 knockdown partially rescued neuronal migration into the CP. (c) Overexpression of RNAi-resistant NDEL1 or NDE1 with NDEL1 knockdown rescued the neuronal migration into the CP, even increasing the fraction of electroporated cells in the CP. Data are presented as mean±s.e.m., and unpaired t-tests used for all comparisons, *P<0.05, n=3 embryonic brains from different mothers. Scale bar, 50 µm.

Fig 3: Roles of NDE1 and NDEL1 in the neural progenitor cell cycle.Our data indicate that NDE1 is required for three distinct non-mitotic processes in the radial glia progenitor (RGP) cell cycle: G1-to-S progression (1), apical INM during late G2 (2) and G2-to-M transition (3). NDEL1 overexpression rescues (1) and (2), but not (3). Interference with the RGP cell cycle at any or all of these stages is proposed as an important contributor to microcephaly. Scale of image does not reflect actual duration of cell cycle stages.

Fig 4: (A) FLAG–DISC1 co-immunoprecipitate V5–NDE1–SSSC from COS7 lysates. (B) Antibody NDE1 93 co-immunoprecipitates endogenous NDE1 and DISC1 from SH-SY5Y lysates. Arrowheads indicate previously observed DISC1 isoforms. (C) GFP–NDE1–sssc co-immunoprecipitate V5–DISC1 when both proteins are in vitro transcribed and translated. GFP–NDE1 denote absence of GFP–NDE1 in the co-immunoprecipitation reaction. (D) V5–NDE1–SSSC and FLAG–DISC1 co-localise at centrosome-like structures in HEK293T cells, marked with an arrow.

Fig 5: (A) Overlapping expression of exogenous NDE1–SSSC and ?-tubulin in HEK293T cells. An arrow marks the centrosome. (B) The 92 and 93 antibodies detect GST–NDE1–SSSC, but not GST–NDEL1–PLSV. NDEL1 231 detects primarily GST–NDEL1. Three commercial NDE1 antibodies ab57430 (Abcam), H00054820-M01 (Abnova) and 10233-1-AP (PTG) all cross react with GST–NDEL1. (C) Antibodies 92 and 93 were incubated with (+) or without (-) their antigenic peptide over-night and then used to stain a GST–NDE1 Western blot. In both cases, pre-absorption reduced the antibody signal. (D) Both 92 and 93 detect V5–NDE1 when over-expressed in COS7 and Western blotted. (E) Both the 92 and 93 antibodies co-localise with a V5 antibody when V5–NDE1–SSSC is over-expressed in COS7 cells. Co-localisation is at large puncta, commonly seen when NDE1 is over-expressed in this cell line, but unrepresentative of endogenous NDE1 localisation. (F) NDE1 92 and 93 antibodies detect a species of a size similar to the predicted ~38 kDa in SH-SHY5Y lysates. (G) NDE1 localises to the centrosome in SH-SY5Y cells, marked with an arrow. (H) Large nuclear puncta are seen when V5–NDE1 is over-expressed in SH-SY5Y cells. This is detectable using both the 93 and anti-V5 antibodies. (I) Endogenous NDE1 co-immunoprecipitates endogenous LIS1 from COS7 lysates. (J) Endogenous NDE1 and LIS1 co-localise at a centrosome-like structure in SH-SY5Y cells. A likely centrosome is marked with an arrow.