Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors

G protein β subunits perform essential neuronal functions as part of G protein βγ and Gβ(5)-RGS (Regulators of G protein Signaling) complexes. Both Gβγ and Gβ(5)-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a co-chaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesis in vivo, we deleted the Phlp1 gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a more than 10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ(5) and RGS9-1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstrate in vivo that PhLP1 is required for the folding and assembly of both Gβγ and Gβ(5)-RGS9

Lamellipodia are crucial for haptotactic sensing and response

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis – differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments

Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling.

G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling

Lamellipodia are crucial for haptotactic sensing and response

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis – differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments

Cone transretinal ERG parameters.

<p>The following parameters are from the fits of the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117129#pone.0117129.g006" target="_blank">Fig. 6</a>. R_max, maximal response amplitude</p><p>time-to-peak (T_peak) and integration time (T_integr.) refer to responses whose amplitudes were < 0.2 R_max and fell within the linear range</p><p>S_f(n), normalized dim flash fractional sensitivity (amplitude of dim flash response divided by flash strength and then normalized for the amplitude of saturating response)</p><p>I_1/2, half-saturating light intensity</p><p>n (I_1/2), Hill coefficient in the Naka-Rushton equation</p><p>τ_rec, time constant of single-exponential decay of dim flash response recovery phase. Values are means ± SEM. <i>NS</i> (not significant) indicates <i>p</i> > 0.05 and *** indicates <i>p</i> < 0.001, all compared to <i>PhLP</i>^<i>+/+</i><i>Cre</i>^<i>+</i><i>Gnat1</i>^<i>-/-</i> values.</p><p>Cone transretinal ERG parameters.</p

ERG analysis of cone-specific PhLP1-deleted mice.

<p>A-B) Families of ERG responses for <i>PhLP1</i>^<i>+/+</i><i>Cre</i>^<i>+</i> and <i>PhLP1</i>^<i>F/F</i><i>Cre</i>^<i>+</i> mice under photopic (A) and scotopic (B) conditions. Light intensity values are in log candela seconds per square meter. C) Intensity-response relationships for photopic b-waves (n = 8). Data were fit to the Naka-Ruston function that yielded the parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117129#pone.0117129.t001" target="_blank">Table 1</a>. D-E) Intensity-response relationships for scotopic a-waves (D) and scotopic b-waves (E) (n = 4). Data were fit to a double hyperbolic function (30). All data are means ± SEM.</p

Protein expression in retina from PhLP1-deleted cones.

<p>A) Immunoblots of whole-retinal extracts for PhLP1, cone G_t2 subunits, RGS9-1, Gβ₅, M-opsin, cone arrestin, and rod G_t1 subunits. B) Quantification of immunoblot bands from panel A relative to the wild-type. All data are means ± standard error of the mean (SEM) from 3–6 mice.</p

Immunolocalization of RGS9-1 and Gβ₅ in PhLP1-deleted cones.

<p>Retinal cross-sections from <i>PhLP1</i>^<i>+/+</i><i>Cre</i>^<i>+</i> and <i>PhLP1</i>^<i>F/F</i><i>Cre</i>^<i>+</i> mice expressing EGFP in cones were probed with antibodies to RGS9 (A) or Gβ₅ (B) and detected with AF555-conjugated secondary antibody fluorescence (red). EGFP fluorescence (green) marks the cones. The red and green signals were merged to show expression of RGS9 and Gβ5 in cones. White arrows highlight one of several cone outer segments in these views that express RGS9 or Gβ5 and EGFP.</p

Immunolocalization of G_t2 subunits in PhLP1-deleted cones.

<p>A) Retinal cross-sections from <i>PhLP1</i>^<i>+/+</i><i>Cre</i>^<i>+</i> and <i>PhLP1</i>^<i>F/F</i><i>Cre</i>^<i>+</i> mice were probed with antibodies specific to Gα_t2, Gβ₃, Gγ_c, and cone M-opsin and detected with FITC-conjugated secondary antibodies (red).</p

ERG parameters for photopic b-waves.

<p>The following parameters are from the fits of the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117129#pone.0117129.g005" target="_blank">Fig. 5C</a>. R_max, maximal response amplitude</p><p>I_1/2, half-saturating light intensity. Values are means ± SEM. <i>NS</i> (not significant) indicates <i>p</i> > 0.05 and ** indicates <i>p</i> < 0.005, all compared to <i>PhLP1</i>^<i>+/+</i><i>Cre</i>^<i>+</i> values.</p><p>ERG parameters for photopic b-waves.</p