Assembly of the dynamin GTPase Drp1 into constriction-competent oligomers is a key event in mitochondrial division. Here, Ji et al. show that Drp1 oligomerization can occur on endoplasmic reticulum through an ER-bound population of the tail-anchored protein Mff.

Intracellular protein gradients underlie essential cellular and developmental processes, but the mechanisms by which they are established are incompletely understood. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 forms an anterior-rich cytoplasmic gradient that causes the RNA-binding protein POS-1 to form an opposing, posterior-rich gradient. We demonstrate that the polo-like kinase PLK-1 mediates the repulsive coupling between MEX-5 and POS-1 by increasing the mobility of POS-1 in the anterior. PLK-1 is enriched in the anterior cytoplasm and phosphorylates POS-1, which is both necessary and sufficient to increase POS-1 mobility. Regulation of POS-1 mobility depends on both the interaction between PLK-1 and MEX-5 and between MEX-5 and RNA, suggesting that MEX-5 may recruit PLK-1 to RNA in the anterior. The low concentration of MEX-5/PLK-1 in the posterior cytoplasm provides a permissive environment for the retention of POS-1, which depends on POS-1 RNA binding. Our findings describe a novel reaction/diffusion mechanism in which the asymmetric distribution of cytoplasmic PLK-1 couples two RNA-binding protein gradients, thereby partitioning the cytoplasm.

Drpl is a dynamin GTPase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51 and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is ER-associated in mammalian cells, and is distinct from mitochondrial or peroxisomal Drp1 populations. Sub-populations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, ER-bound) and mitochondrial division, while Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.SummaryAssembly of the dynamin GTPase Drp1 into constriction-competent oligomers is a key event in mitochondrial division. Here, Ji et al show that Drp1 oligomerization can occur on endoplasmic reticulum through an ER-bound population of the tail-anchored protein Mff.Abbreviations used in this paper: Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; INF2, inverted formin 2; KD, siRNA-mediated knock down; KI, CRISPR-mediated knock in; KO, CRISPR-mediated knock out; LatA, Latrunculin A; MDV, mitochondrially-derived vesicle; Mff, mitochondrial fission factor; MiD49 and MiD51, mitochondrial dynamics protein of 49 and 51 kDa; OMM, outer mitochondrial membrane; TA, tail-anchored.

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3′UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans. Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3′UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

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