NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75NTR collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75NTR both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75NTR. When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75NTR, was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75NTR partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance

A Developmentally Regulated Kinesin-related Motor Protein from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive α-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end–directed microtubule motility in vitro at a speed of 0.14 μm/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells

Association of NGF receptors and cytoskeletal elements with DRMs under different experimental conditions.

<p>A) Western blots showing TrkA and p75^NTR in floating DRMs prepared after 10 min NGF treatment and using nuclease (Benzonase) rather than sonication to break up nucleic acids prior to equilibrium density gradients. Western blots of flotation equilibrium gradients of detergent-resistant fraction were probed with anti-TrkA, -pTrkA, -SHP-1, -p75^NTR, and -tubulin (indicated). Blots include the detergent-sensitive (P1M) fraction and size standards (S) to the left of DRM gradient fractions. B) Left: ¹²⁵I-NGF in DRM without (open squares) and with (closed circles) in vitro reactions with ATP. Right: Quantification of chemiluminescent signals from western blots is compared to ¹²⁵I-NGF for Benzonase-treated samples as in A. Fraction number is plotted on the x-axis of plot on the left; fraction 1 has the highest density. Signals from western blots were quantified and plotted vs. density together with ¹²⁵I-NGF (closed circles) on the right. The y-axis for TrkA (closed squares), p75^NTR (open circles), SHP-1 (open squares), and tubulin (closed triangles) is arbitrary units (chemiluminescent pixel volume). C) Western blots showing actin in floating DRMs prepared as in A, with and without in vitro reactions with ATP (–, +ATP). Data obtained under these conditions for TrkA, tubulin, and actin were quantified and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>. After 10 min internalization, in vitro reactions with ATP caused a significant increase of TrkA (p<0.01) and NGF and tubulin (p<0.001) in floating DRMs. A decrease in actin (+ATP) was noted but was not statistically significant.</p

TrkA and p75^NTRin endosomes and endosomal DRMs.

<p>A) Endosomes from cells treated 10 min with NGF. Organelles that emerged from cells mechanically permeabilized by a single pass through a Balch homogenizer were subjected to velocity sedimentation followed by equilibrium flotation as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Mccaffrey1" target="_blank">[7]</a>. Shown is the flotation equilibrium gradient from velocity gradient fraction 3, which contains TrkA and p75^NTR endosomes (indicated) that floated to their equilibrium density. Blots were probed with anti-pTrkA, -p75^NTR, and -SHP-1 (indicated). B) The detergent-resistant fraction containing endosomes from untreated or NGF-treated cells before (no reaction) or after in vitro reactions (+ATP) was fractionated on iodixanol velocity gradients as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Maccormick1" target="_blank">[34]</a>. Pools from the bottom of the gradient containing microtubules (MT) and control samples from the top of the gradient (C) were collected for immunoprecipitations with anti-tubulin (MTIP). One-ninth of each sample was TCA precipitated before immunoprecipitation (input). Western blots were probed with anti-TrkA and anti-p75^NTR (indicated). pTrkA was not detected in endosomal DRMs (not shown).</p

Amount of Radioactive Ligands Associated With Cell Fractions.

<p>Cells were bound to radiolabelled ligand, washed, and subjected to internalization 10 min at 37°C. Mechanical permeabilization, fractionation, and detergent extraction was performed exactly as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Grimes1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Grimes2" target="_blank">[36]</a>.</p

NGF associates with lipid rafts before and after initiation of membrane traffic and signal transduction.

<p>¹²⁵I-NGF was bound to PC12 cells in the cold, the cells were washed and warmed for the indicated periods of time. 0 min represents cells bound to NGF but not warmed. Flotation equilibrium iodixanol gradients were performed using sonication to resuspend the detergent-resistant fraction. A) Plots of DRM gradients after pulse-stimulation with ¹²⁵I-NGF for 0, 2, 10, and 30 min. Refractive index measurements were taken on each fraction and converted into density using a formula derived empirically (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#s4" target="_blank">Methods</a>); density is plotted on the x-axis. There also was non-floating NGF in the detergent-resistant pellet, whose distribution in fractions of higher density is consistent with diffusion in a bottom-loaded sample. B, C) Amount of NGF and density of floating DRMs. The amounts of ¹²⁵I-NGF in the floating DRM peak containing ¹²⁵I-NGF were quantified and compared to amounts in detergent-soluble membranes and other fractions (B, %WC=percent of whole cell). Amounts in the floating DRM peak are plotted as the percent in the whole cell. A transient increase in the density of the floating ¹²⁵I-NGF DRM peak was noted after 2 and 10 min (C). A higher density suggests a higher protein:lipid ratio. Error bars are SEM. D) DRM fraction from rat dorsal root ganglia neurons bound to ¹²⁵I-NGF and warmed for 10 min as above. The floating peak had a slightly higher density (1.18 g/ml) than that in PC12 cells (1.16 g/ml).</p

In vitro reactions cause microtubules to associate with lipid rafts, attracting NGF and TrkA but excluding p75^NTR.

<p>Cells were bound to NGF and sonicated DRMs fractionated on flotation equilibrium gradients as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g001" target="_blank">Figure 1</a> (0 min internalization). A) Western blots with anti-TrkA, -p75^NTR, -tubulin, and -flotillin (indicated) from cells fractionated before (–ATP) and after (+ATP) in vitro reactions with ATP. Gradients were collected from the bottom, so lower numbered fractions have higher density. The floating DRM peak is in the middle of the gradients. B) The plot in the lower left is ¹²⁵I-NGF in DRMs from cells before (closed diamonds) or after (open circles) in vitro reactions with ATP. The amounts of ¹²⁵I-NGF, and proteins shown in A in the floating DRM peak coincident with ¹²⁵I-NGF, were quantified and compared to amounts in detergent-soluble membranes and other fractions. Amounts in the floating DRM peak are plotted as the percent in the whole cell under these conditions (–ATP, +ATP). Error bars are SEM. In vitro reactions with ATP caused a significant increase of TrkA (p<0.01) and NGF and tubulin (p<0.001), and a decrease in p75^NTR (p<0.05) in floating DRMs after 0 min internalization.</p

NGF differently affects association of TrkA and p75^NTRwith floating DRMs.

<p>A) Floating DRMs were isolated using Benzonase treatment after 10 min without (open diamonds) and with (closed squares) NGF treatment without (no rxn, left) and with (+in vitro rxn, right) subsequent in vitro reactions with ATP. TrkA (upper panels) and p75^NTR (lower panels) were quantified from western blots probed simultaneously in the same antibody solution, exposed for the same amount of time, for all conditions. Data are plotted using the same y-axis (chemiluminescence for TrkA and p75^NTR) for all conditions. In vitro reactions had little influence on the amounts of TrkA in floating DRMs in the absence of NGF, but increased TrkA in DRMs in the presence of NGF. In vitro reactions caused p75^NTR to increase in the floating peak in the absence of NGF, but decrease in the presence of NGF. B) Amounts of TrkA, p75^NTR, tubulin, and flotillin in the floating DRM peak prepared using sonication as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>, plotted as the percent of the whole cell for control (–NGF) and NGF-treated (+NGF) for cells subjected to in vitro reactions. Under these conditions, NGF caused an increase in TrkA (p<0.1) and tubulin (p<0.05) in floating DRMs, yet caused a significant decrease (p<0.05) in p75^NTR in floating DRMs.</p

TrkA is bound to microtubules in floating DRMs. Floating DRMs were isolated after 10

<p> <b>min NGF treatment and in vitro reactions.</b> A) Microtubules were immunoprecipitated from the floating peak with anti-β-tubulin and western blotted for TrkA (upper panel) and tubulin (lower panel). B) In vitro reactions without (ATP only) or with biotinylated tubulin added during the last 5 min of the reaction (+biotin-tubulin) were performed. The floating DRM peak was immunoprecipitated with streptavidin or anti-TrkA (indicated) and western blotted for anti-TrkA (upper panel) and anti-biotin (lower panel). p75^NTR was not reproducibly detected in microtubule immunoprecipitations from DRMs under these conditions. C, D) Images of permeabilized cells after in vitro reactions with biotinylated tubulin added during the last 5 min of the reaction. Texas red streptavidin stained discreet foci at or near the plasma membrane, shown in a group of permeabilized cells in C and an individual cell in D.</p