Panel E shows DRG growth cones stained to label F-actin (Alexa Fluor 488-phalloidin, green) or free actin barbed ends (Rh-actin, red). features, as shown in sample kymographs. NIHMS216897-supplement-Supp_Fig_s2.tif (927K) GUID:?C54C8EBD-3012-4768-8F9D-15AC2508A88B Supp Fig s3: Physique S3 A micropipette coated with nitrocellulose and protein establishes a diffusible gradient. A micropipette was coated with nitrocellulose and 1 g/ml rhodamine-BSA (A) or rhodamine-BSA complexed with Chariot reagent (B) and lowered into a dish with medium. Fluorescent images were taken after 1, 5, 10, and 15 min. The concentric gradients were measured using the linescan function in Metamorph. Insets show the gradients between 68C102m from your micropipette tip. NIHMS216897-supplement-Supp_Fig_s3.tif (3.1M) GUID:?6EDFF742-1A0F-4BE1-A2D0-78D9CC88F1CA Supp Fig s4: Physique S4 Expressing constitutively-active LIMK (CA-LIMK) or ADF hRNA results in decreased AC activity. A) Dissociated DRGs were transfected with GFP or GFP + CA-LIMK, cultured 24 hr, fixed and stained with pAC. Bar graph shows mean growth cone pAC intensities in GFP- and CA-LIMK-transfected growth cones. B) Western blot of extracts from chick embryo fibroblasts infected with hRNA-expressing adenoviral vectors for the time indicated before preparing cell extracts. Control vector expresses a rat hRNA that does not silence the chick protein. Two different loading volumes of the 120 hr extracts are shown for the chick ADF silencing vector. Contamination efficiency for these chick fibroblasts is about 80% (based on GFP co-expression from your virus) suggesting silencing at 72 hr is about 65% and at 120 hr is about 80%. C) DRGs transfected with ADF hRNA, cultured 48hr, then fixed and stained with pAC (reddish). Note the level of anti-pAC staining of the adjacent non-transfected cell. Scale BRL 52537 HCl bars, 10 m. Statistical significance using Students test. Data are means SEM; *P 0.05. NIHMS216897-supplement-Supp_Fig_s4.tif (3.9M) GUID:?873A31E9-5860-46EA-99ED-CA399A694C0B Supp Movie s1: Movie S1 A nitrocellulose-coated micropipette incubated with netrin-1 induces attractive turning when lowered to one side of an extending retinal growth cone. Video shows one frame every 2 min, for a total period of 78 min. NIHMS216897-supplement-Supp_Movie_s1.mov (340K) GUID:?FC3C1062-C31B-4C1D-8461-907EA016E623 Supp Movie s2: Movie S2 A nitrocellulose-coated micropipette incubated with Chariot-A3 complexes induces attractive turning when lowered to one side of an extending DRG growth cone. Video shows one frame every 2 min, for a total period of 38 min. NIHMS216897-supplement-Supp_Movie_s2.mov (480K) GUID:?B9BF257A-0942-4EE6-ADE2-B940D56EEB8B Supp Movie s3: Movie S3 A nitrocellulose-coated micropipette incubated with Chariot-A3 complexes induces attractive turning when lowered to one side of an extending retinal growth cone. Video shows one frame every 2 min, for a total period of 64 min. NIHMS216897-supplement-Supp_Movie_s3.mov (251K) GUID:?979D5305-8F57-4FA3-8171-19C43420F346 Abstract Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues Goat monoclonal antibody to Goat antiMouse IgG HRP. change the cytoskeleton to guide growth cone pathfinding. Here we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is usually decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin BRL 52537 HCl levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side made up of higher AC activity. (Gunderson, 1985; Gunderson and Barrett, 1979; Letourneau, 1978) and (Glebova and Ginty, 2004; Hassankhani et al., 1995; Menesini Chen et al., 1978; Patel et al., 2000) show that this neurotrophin nerve growth factor (NGF) is an attractive cue for sensory and sympathetic neurons and is necessary for target BRL 52537 HCl innervation. Netrins are another well-characterized group of guidance cues that induce attraction or repulsion depending on receptor expression (Kennedy et al., 1994; Moore et al., 2007; Round and Stein, 2007). It is proposed that attractive cues, such as NGF and netrin-1, favor actin stabilization and polymerization in the growth cone proximal side (Dent et al., 2004; Gallo and Letourneau, 1997, 2000; Lebrand et al., 2004; Lin and Forscher, 1993; OConnor and Bentley, 1993; Quinn et al., 2008; Sabry et al., 1991;.