Forces Planar to the Membrane

Testing the notion of myo1c remaining anchored in the plasma membrane while experiencing forces in plane with the membrane is essential to identifying the possible

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functions of myosin-Is (Figure 50). For myo1c to be functional as a motor while bound to the membrane, it must be stationary at forces up to 2 pN, the estimated force of a myo1c powerstroke (Gillespie and Cyr 2004). We can estimate the minimum velocity of an individual powerstroke of myo1c by dividing the powerstroke distance of 4.2 nm by the lifetime of actin attachment 50 ms (Gillespie and Cyr 2004) to get a velocity of 0.084 µm/s. To withstand forces of up to 2 pN, the drag coefficient of myo1c on the membrane would have to be greater than 2.4 x 10-11 N·s/µm in accordance with:

Equation 10

𝐹 = 𝑣𝛾

where F is the force against the plane of the membrane, v is the velocity of the powerstroke, and γ is the drag coefficient. Using the Einstein relation,

Equation 11

𝐷 =𝑘𝑇 𝛾

where D is the lateral diffusion coefficient, and kT is the Boltzman constant at 37 °C, 4.11 x 10-15 N·µm, this would require the lateral diffusion coefficient of a myo1c bound to the plasma membrane to be less than 1.7 x 10-4 µm2/s.

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Figure 50 Forces planar to the membrane

Figure 50: Cartoon of myo1c producing forces planar to the membrane. A) Myo1c bound to both actin and PIP2 pre-powerstroke. B) Myo1c bound to the membrane remains stationary as the actin filament is translated relative to the membrane after a powerstroke. C) Actin filament remains stationary as myo1c bound to PIP2 laterally diffuses in the membrane after a powerstroke.

158 4.4.1.1 Lateral Diffusion

A diffusion coefficient of 1.7 x 10-4 µm2/s is much slower than the diffusion rates measured for many lipids (6 µm2/s) (Scandella, Devaux et al. 1972) and integral membrane proteins (0.5-2 µm2/s) (Snapp 2003). Diffusion rates of phosphatidyl inositol associated proteins, such as PLCδ-PH domain, have been measured using FRAP to be close to cytoplasmic diffusion rates (3.5 µm2/s); however, no controls for the possibility of the dissociation of bleached proteins from the membrane and association of unbleached protein were shown (Brough, Bhatti et al. 2005). Additionally, it was found for eGFP-PH123 that the mean-squared deviation in position did not increase with time and remained close to the limit of position detection (0.03 µm2) (Mashanov, Tacon et al. 2004).

Interestingly, the diffusion rate for PIP2 was recently measured to be as low as 3.9 e-4 µm2/s in mouse arterial myocytes (Cho, Kim et al. 2005). The diffusion rate was found to be dependent on the cytoskeleton and is ten thousand times slower than other phosphatidyl inositols measured in the same study. PIP2 with a lateral diffusion coefficient of 3.9 e-4 µm2/s would provide the approximate resistance for a myo1c motor to translate an actin filament in relation to the plane of the membrane during a powerstroke.

There are many possibilities that could account for the five orders of magnitude difference in lateral diffusion rates between PIP2 and other lipids. It has been suggested that lipids can segregate into microdomains created by mismatches in hydrophobic

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thickness, van der Waals interactions, and acyl chain entropy (Marguet, Lenne et al. 2006). Thus, PIP2 could be segregated into these microdomains which may have a lower overall diffusion rate than the rest of the membrane. Although PIP2 has a polyunsaturated chain and should not partition into microdomains without other factors acting on it (McLaughlin, Wang et al. 2002), studies have shown that some microdomains are enriched in PIP2 (Golub, Wacha et al. 2004).

A possibility that also accounts for the dependence on the cytoskeleton is that the lateral diffusion of PIP2 could be limited by the mesh size of the cytoskeleton, a model commonly referred to as the picket fence model (Sako and Kusumi 1995). In this model, it is suggested that the actin cytoskeleton acts as a fence that encloses a domain of lipids and lipid-bound proteins. Transmembrane proteins that are associated with the cytoskeleton act as pickets in the membrane and further limit the mobility of lipids from diffusing outside of these domains. The actin cytoskeleton lies close to the plasma membrane at just 10.2 nm (Morone, Fujiwara et al. 2006). The mesh sizes created by the actin network differ from cell types, ranging from 0.0027 µm2 in FRSK cells and to 0.039 µm2 in NRK cells. These measurements correlate well with the measured lateral diffusion of a fluorescently labeled phospholipid used in the same study, which diffused within a compartment of 0.0021 µm2 in FRSK cells and 0.043 µm2 in NRK cells (Morone, Fujiwara et al. 2006). However, this model alone cannot explain the five magnitude difference in lateral diffusion rates of PIP2 to other phospholipids. Incorporating the findings that so many proteins bind to PIP2 and not to

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other phospholipids might be enough to explain the lower diffusion rates. The lateral diffusion rates of these proteins would be limited by the cytoskeleton, thus limiting PIP2 bound to them, while phospholipids not bound to proteins would be free to diffuse.

An alternative explanation is that the low lateral diffusion rates observed for PIP2 may be the effect of myo1c binding rather than the cause of myo1c having a low lateral diffusion rate while it is bound to PIP2. The most likely explanation for the five magnitude lower diffusion rate of PIP2 to other phospholipids is a direct link between PIP2 and the cytoskeleton. It has been shown that PIP2 levels regulate membrane dynamics and cell shape by locally increasing and decreasing the interactions between the plasma membrane and actin cytoskeleton (Raucher, Stauffer et al. 2000). The low diffusion rate of PIP2 could be the effect of myo1c or another protein linking the phosphatidyl inositol directly to the cytoskeleton. If PIP2 is linked to the cytoskeleton through a protein tether, it would significantly inhibit its lateral diffusion, which would explain the large difference between lateral diffusion rates between PIP2 and other phospholipids. With the cytoskeleton being within 10.2 nm of the plasma membrane (Morone, Fujiwara et al. 2006), it is plausible that myo1c may function as a protein tether, especially given that a similar myosin-I, myo1a, has an estimated length of 22 nm (Jontes and Milligan 1997). However, PIP2 would not be able to anchor myo1c to the membrane if myo1c is the reason for the lateral diffusion rate of 3.9 x 10-4 µm2/s.

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