Myo1c binds tightly and specifically to phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate

Abstract

Myosin-I is the single-headed member of the myosin superfamily that associates with acidic phospholipids through its basic tail domain. Membrane association is essential for proper myosin-I localization and function. However, little is known about the physiological relevance of the direct association of myosin-I with phospholipids or about phospholipid headgroup-binding specificity. To better understand the mechanism of myosin-I-membrane association, we measured effective dissociation constants for the binding of a recombinant myo1c tail construct (which includes three IQ domains and bound calmodulins) to large unilamellar vesicles (LUVs) composed of phosphatidylcholine and various concentrations of phosphatidylserine (PS) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). We found that the myo1c-tail binds tightly to LUVs containing >60% PS but very weakly to LUVs containing physiological PS concentrations (<40%). The myo1c tail and not the IQ motifs bind tightly to LUVs containing 2% PIP(2). Additionally, we found that the myo1c tail binds to soluble inositol-1,4,5-trisphosphate with nearly the same affinity as to PIP(2) in LUVs, suggesting that myo1c binds specifically to the headgroup of PIP(2). We also show that a GFP-myosin-I-tail chimera expressed in epithelial cells is transiently localized to regions known to be enriched in PIP(2). Our results suggest that myo1c does not bind to physiological concentrations of PS but rather binds tightly to PIP(2).

Fig. 1.

Association of the myo1c-tail with LUVs. (A) SYPRO-red stained SDS–polyacrylamide gel of myo1c-tail from the pellets of a sedimentation assay with 0–400 μM LUVs (total lipid) containing 2% PIP₂. The last three lanes are myo1c-tail standards used for normalization. (B) Lipid concentration dependence of 40 nM myo1c-tail binding to LUVs composed of PC and (○) 0% PS, (□) 20% PS, (◇) 40% PS, (▴) 60% PS, (▾) 80% PS, and (•) 2% PIP₂. Each point is the average of two to six measurements. The solid and dashed curves are the best fits of the 60% PS and 2% PIP₂ data to hyperbolae, respectively. The K_eff of each data set is listed in Table 1.

Fig. 2.

Binding of 40 nM myo1c-tail to 30 μM LUVs (total lipid) composed of 0–10% PIP₂. The percent of membrane-bound myo1c-tail is plotted as a function of the percentage of PIP₂ in the LUVs rather than total lipid concentration. Each point is the average of three measurements. (Inset) The same data plotted as a function of the accessible PIP₂ concentration (○). Data in Inset also include the percent of 40 nM myo1c-tail bound to (▵) 60 μM LUVs containing 0–5% PIP₂ and (•) 0–400 μM LUVs containing 2% PIP₂ from Fig. 1B. The solid line is the best fit of all of the data to a hyperbola, yielding K_eff = 0.31 ± 0.06 μM.

Fig. 3.

The myo1c-tail binds InsP₃. (A and B) Gel filtration elution profiles of samples containing 10 μM PLCδ-PH (A) or 10 μM myo1c-tail (B) in the presence of excess ³H-Ins(1,4,5)P₃. The concentrations of InsP₃ (solid lines) and PLCδ-PH or myo1c-tail (dotted lines) are shown. InsP₃ run in the absence of protein in a separate experiment is also shown (dashed line) as a reference elution profile. (C) Binding of 100 nM myo1c-tail to 60 μM LUVs containing 2% PIP₂ in the presence of 0–10 μM PLCδ-PH. (Inset) A SYPRO-red-stained SDS–polyacrylamide gel showing a LUV-bound (top) myo1c-tail and (bottom) PLCδ-PH as a function of total PLCδ-PH concentration. (D) Binding of 100 nM myo1c-tail to 60 μM LUVs containing 2% PIP₂ in the presence of 0–10 μM InsP₃. (Inset) A SYPRO-red-stained SDS–polyacrylamide gel showing a LUV-bound myo1c-tail as a function of total InsP₃ concentration. Solid lines are fits to a competition binding equation, yielding K_eff = 0.30 ± 0.03 μM for PLCδ-PH and K_d = 0.072 ± 0.12 μM for InsP₃. Error bars represent ± 1 standard deviation (n = 6).

Fig. 4.

The effect of calmodulin and calcium on myo1c-binding LUVs. (A) Forty nanomolar myo1c-tail binding to 60 μM LUVs containing (•) 2% PIP₂ or (▴) 60% PS LUVs in the presence of 0–100 μM calmodulin. Each point is the average of two measurements. (B) Forty nanomolar myo1c-tail binding to 0–400 μM LUVs containing 2% PIP₂ in the (•) absence and (○) presence of 10 μM free calcium. The solid line is the best fit to a rectangular hyperbola. Error bars represent ± 1 standard deviation (n = 4–6). (C) Lipid concentration dependence of 40 nM myo1c–motor–IQ binding to LUVs composed of PC and 20% PS (■, □) or 2% PIP₂ (•, ○) in the absence (closed symbols) or presence (open symbols) of 10 μM free calcium.

Fig. 5.

Cellular distribution of GFP–myo1c-tail during influx of calcium. Fluorescence micrographs of a transfected normal rat kidney epithelial cell showing GFP fluorescence before, during, and after incubation with 10 μM ionomycin in medium containing 1.2 mM CaCl₂. (Scale bar, 15 μm.) See Movie 1, which is published as supporting information on the PNAS web site, for movie sequence.

Fig. 6.

Cellular distribution of GFP–myo1c-tail during macropinocytosis. Fluorescence micrographs of a transfected NRK cell showing GFP fluorescence around a newly internalized macropinosome (arrow). Note the loss of fluorescence around macropinosome after it is internalized. (The time stamp is min:sec, and the scale bar is 5 μm.) See Movie 2, which is published as supporting information on the PNAS web site, for movie sequence.