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Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis

Erik A. Lundquist^1,*,, Peter W. Reddien^2,*, Erika Hartwieg², H. Robert Horvitz² and Cornelia I. Bargmann³

¹ Department of Molecular Biosciences, University of Kansas, 5049 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
² Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
³ Howard Hughes Medical Institute, Department of Anatomy Box 0452, University of California, San Francisco, CA 94143, USA
^* These authors contributed equally to this work

View larger version (76K): [in a new window]   Fig. 1. The C. elegans Rac gene family. Alignment of the C. elegans Rac-like proteins CED-10 (Genbank Accession, AAF33846), RAC-2 (CAB05247), RAC-3 (CAB05244) and MIG-2 (AAC47729) with human (Hs) Rac1 (NP008839). Gray shading indicates residues that are identical in at least three out of five sequences. The myristylation signal (myr) of MIG-2 and motifs conserved in all five molecules are underlined. The P-loop region interacts with phosphoryl groups of GDP and GTP, the switch regions change conformation upon GDP/GTP exchange, and the C-terminal membrane targeting signal typically results in prenylation (Bourne et al., 1991; Zhang and Casey 1996). The glycine 12 (G12) residue (G16 in MIG-2) that is altered to a valine in constitutively-active alleles of Rho-family GTPases including mig-2(rh17) is indicated by a V above the alignment. The G12 residue was altered to a valine in the ced-10(G12V) cDNA construct. A diagram illustrating the identity relationships among the molecules is shown below the alignment.

View larger version (103K): [in a new window]   Fig. 2. CAN axon defects in ced-10(M+); mig-2 double mutants. (A) A ced-10(n1993)/dpy-13(e184); mig-2(mu28) animal. This animal was non-Unc, non-Wit, non-Egl and non-pVul. (B) A ced-10(n1993M+); mig-2(mu28) homozygote. This animal was Unc, Dpy, Wit, Egl and pVul. (C-G) Fluorescence micrographs of GFP expression from a ceh-10::gfp transgene expressed in the CAN neurons of living animals. (C) The CAN neuron of a wild-type animal is shown. The out-of-focus bright spot near the vulva is the CAN cell body on the other side of the animal. Some head neurons also express ceh-10::gfp. (D) A ced-10(n1993M+); mig-2(mu28) double mutant. The posterior CAN axon terminated prematurely. CAN cell position was normal in this animal. (E) The posterior CAN axon of a ced-10(M+); mig-2 double mutant displayed an ectopic axon branch. (F) A posterior CAN axon of a ced-10(n1993M+); mig-2(mu28) double mutant was misguided, turned 180° and extended anteriorly before terminating. Arrowheads trace the trajectory of the axon. The background fluorescence is autofluorescence of the gut. (G) A ced-10(n1993M+); mig-2(mu28) double mutant. The CAN cell body failed in its posterior migration. The posterior CAN axon of this animal extended to its normal position in the tail of the animal. The trajectory of the main axon is traced by arrowheads. Anterior, left; dorsal, top. The location of the vulva is indicated by a V. Scale bars: 50 µm in A-D,G; 10 µm in E,F.

View larger version (19K): [in a new window]   Fig. 3. mig-2, ced-10, and rac-2/3 redundantly and cell-autonomously control CAN axon pathfinding and cell migration. CAN axon pathfinding and cell migration were scored in living animals using ceh-23::gfp integrated transgenes (see Materials and Methods). The percentages of animals displaying defective posterior CAN axon pathfinding and defective CAN cell migration for each genotype are shown. The M+ designation in the genotypes indicates that animals had a wild-type maternal contribution of gene function. ‘+’ animals carried the kyIs8 integrated ceh-23::gfp transgene. lqEx27 is an extrachromosomal array bearing the unc-115 promoter::ced-10(G12V) cDNA transgene. lqEx53 is an array bearing the ceh-10 promoter::mig-2(+) cDNA transgene. lqEx56 is an array bearing the ceh-10 promoter::ced-10(+) cDNA transgene. Genotypes described as ‘sib’ represent the non-array-bearing siblings from the same brood as array-bearing animals. Error bars define 95% confidence intervals of the standard error of the proportion.

View larger version (63K): [in a new window]   Fig. 4. mig-2 and ced-10 redundantly control the development of the dorsal and ventral nerve cords and D-class motor axon pathfinding. (A,B) Ventral neurons expressing an unc-25::gfp reporter. Anterior is left; the left side of each animal is upwards. Scale bars: 10 µm. (A) The D class neurons in the ventral cord of a wild-type animal displayed normal fasciculation, cell body positions and commissure extensions. Arrow, cell bodies; arrowheads, ventral cord fascicle; c, commissure. (B) D class neurons in a ced-10(n1993M+); mig-2(mu28) animal. Black arrowheads, terminated and branched axons attempting to form commissures; white arrowheads, defasciculated ventral cord; arrows, D class motoneuron cell bodies. Thick arrow, cell body displaced from the ventral cord. (C) The average number of commissures that reached the dorsal cord was determined from 20 animals for wild type and mutant. Error bars represent standard errors of the mean. (D) An electron micrograph of a wild-type ventral cord. There are 52 axons in the right fascicle and four axons in the left fascicle. Scale bar: 500 nm. (F) An electron micrograph of the ventral cord of a ced-10(n1993M+); mig-2(mu28) double mutant. There are 32 axons in the right fascicle and three axons in the left fascicle. The arrow indicates three axons separated from the main fascicles. Scale bar: 500 nm. (E,G) Tracings of electron micrographs in D,F, respectively. N, nucleus. M, muscle. Red asterisks, axons.

View larger version (125K): [in a new window]   Fig. 5. ced-10 is expressed broadly. Anterior, left; dorsal, top. Scale bars: 5 µm in A; 2.5 µm in B,C; 5 µm in D,E. (A) A mid-L1-stage larva showing GFP::CED-10 expression in all cells examined, including the pharynx, axons of the nerve ring, and neurons along the ventral nerve cord (vnc), where the GABAergic D-class motoneurons reside. GFP::CED-10 localized to the plasma membrane in vnc neuron cell bodies. (B) A different focal plane of the animal in A showing expression of GFP::CED-10 in the hypodermis. The hypodermal seam cells (V cells) are noted with arrows. GFP::CED-10 accumulated at the plasma membrane in these cells. (C) An L2 larva with plasma membrane accumulation of GFP::CED-10 in vnc motoneuron cell bodies. Intestinal expression is also shown. (D) GFP::CED-10 accumulation at the plasma membrane of the CAN neuron cell body in an adult animal. The fused hypodermal seam syncytium also showed plasma membrane-localized GFP::CED-10. (E) An L4 larva with expression of GFP::CED-10 in the posterior distal tip cell of the developing gonad.

View larger version (26K): [in a new window]   Fig. 6. unc-73 and ced-5 but not ced-2 act with rac genes in CAN axon pathfinding and cell migration. Data are displayed as in Fig. 3. The CAN axon pathfinding and cell migration defects of ced-5(n1812) ced-10(n1993) and ced-5(n2002) ced-10(RNAi) mutants (marked by asterisks) were significantly more severe than those of ced-5 and ced-10 mutants alone (P<0.001).

View larger version (36K): [in a new window]   Fig. 7. Models for the redundancy of Rac function in axon pathfinding, cell migration, and cell-corpse phagocytosis. Boxes indicate a required gene and broken arrows indicate a subtle function. (A) mig-2 Rac-like, ced-10 Rac and rac-2/3 Rac act redundantly in CAN axon pathfinding and cell migration. unc-73 Trio acts in all three pathways. ced-5 DOCK180, which acts in parallel with ced-10 Rac, might act with mig-2 Rac-like, rac-2/3 Rac or both (indicated by parentheses). ced-2 CrkII is not involved in CAN axon pathfinding. (B) mig-2 Rac-like, ced-10 Rac and rac-2/3 Rac act redundantly in CAN cell migration. mig-2 Rac-like and ced-10 Rac are both necessary for DTC migration and are regulated by unc-73 Trio, ced-2 CrkII and ced-5 DOCK180. rac-2/3 Rac is not necessary for DTC migration and is not shown here. rac-2/3 Rac is slightly redundant for cell movement with mig-2 but not ced-10. (C) ced-10 Rac is the primary rac involved in phagocytosis, whereas rac-2/3 Rac and mig-2 Rac-like have subtle functions seen only in genetically sensitized backgrounds. unc-73 Trio is not involved in phagocytosis.