Fig. 3: Cdk5 localizes in MAMs and loss of Cdk5 causes increased number and length of ER-mitochondria contact sites and reduced contact distance between these two organelles.

a Immunoblot analysis of purified MAM and mitochondria in wt and Cdk5^−/− MEFs. 50 μl of homogenate (H), crude MAM (C-MAM), purified MAM (P-MAM), and purified mitochondria (MT) were loaded. For subcellular markers, fatty acid-CoA ligase, long-chain (FACL4) and glucose-regulated protein 78 kDa (GRP78) were used for ER-MAM; VDAC for mitochondria-MAM; proliferating cell nuclear antigen (PCNA) for nucleus; and lactate dehydrogenase (LDH) for cytoplasm. Blots shown represent data from one of three independent experiments (n = 3) showing similar results. b–e Transmission electron microscopy (TEM) of ER-mitochondria tethering in wt and Cdk5^−/− MEFs. Images were acquired using a Hitachi H7650 TEM. Scale bar = 100 nm. ER: endoplasmic reticulum; M: mitochondria. Arrowheads are directed at ER-mitochondria contact sites. For the morphometric analysis in c–e, a total of six TEM grids (3.05 mm discs) prepared from two embeddings per genotype were analyzed: four grids from embedding 1 and two grids from embedding 2. Mitochondrial counts and mitochondria-ER contact measurements were performed in groups of two grids (n = 3, where replicates 1 and 2 originate from the four grids from embedding 1 and replicate 3 originates from the two grids from embedding 2. In c, all observed mitochondria in wt and Cdk5^−/− MEFs, irrespective of the number of cells, were counted and the percentage of mitochondria in contact with ER was calculated. In d, the mitochondrial perimeter in contact with ER was measured and the percentage of the total individual mitochondrial perimeter in contact with ER was calculated. In e, distances between ER and mitochondria in contact sites were measured and the percentage of the total contact sites with different ranges of ER-mitochondria distances were calculated. In d and e, a total of at least 40 ER-mitochondria contacts, irrespective of the number of cells, were analyzed per two TEM grids (i.e., at least 120 ER-mitochondria contacts were analyzed per genotype).). Embeddings were from different cultures of MEFs. Values are means ± SEM; n = 3; *p < 0.05 and **p < 0.01 by unpaired Student’s t test.