Regulation of microtubule-based transport by MAP4.
Bottom Line: We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs.We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2-based movement.Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2-dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.
Affiliation: R.D. Berlin Center for Cell Analysis and Modeling and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030.Show MeSH
Mentions: To examine the effect of EGFP-XMAP4 overexpression on MT dynamics, we compared parameters of MT dynamic instability in melanophores induced to aggregate pigment granules overexpressing EGFP-XMAP4 and EGFP by injecting Cy3-tubulin into EGFP-positive cells, tracking tips of growing and shortening MTs, and decomposing tip trajectories into periods of growth, shortening, and pauses. We detected small but significant differences in the parameters of MT dynamic instability between the EGFP-XMAP4- and EGFP-expressing cells (Table 1). In particular, the average lengths of growth and shortening episodes were slightly reduced and the average duration of pauses increased in the melanophores overexpressing EGFP-XMAP4 (Table 1). To determine whether these changes affected the rate of pigment granule aggregation, we used a stochastic computational model for pigment aggregation that we previously developed (Lomakin et al., 2009, 2011). The model computes kinetics of pigment granule aggregation based on the statistics of bidirectional movement of pigment granules, the probability of pigment granule capture by growing MT ends, and parameters of MT dynamic instability and allows one to estimate independent effects of changes in MT dynamics on the pigment aggregation rate (Zaliapin et al., 2005; Lomakin et al., 2009, 2011). The output of the model is the kinetics of gray level decrease, which reflects how fast the cytoplasm becomes increasingly transparent as pigment granules accumulate in the cell center. We first tested the model by performing experimental measurements of gray level decrease in cells expressing EGFP and comparing experimental data with the results of simulations that used the parameters of MT dynamic instability and pigment granule movement measured in these cells (Tables 1 and 2). This comparison showed a close match between the experimental and computed kinetic curves (Supplemental Figure S2). These results, in agreement with our previous analyses (Zaliapin et al., 2005; Lomakin et al., 2009, 2011), demonstrated that the computational model closely reproduced the kinetics of pigment aggregation. We next simulated aggregation of pigment granules using parameters of MT dynamic instability measured in melanophores overexpressing EGFP-XMAP4 (Table 1) without changing the granule movement statistics. These computer simulations generated kinetics of gray level decrease (Figure 3A, open squares) that was close to the kinetics measured for the EGFP-expressing cells (Figure 3A, open circles), with a half-time 4.36 ± 0.19 min. The experimentally measured half-time of gray level decrease estimated for melanophores overexpressing EGFP-XMAP4 (Figure 3A, black squares) was 27.78 ± 4.77 min, much longer than the half-time computed on the basis of dynamic instability parameters. Therefore changes in MT dynamics could not explain the dramatic inhibition of pigment aggregation observed in the EGFP-XMAP4–overexpressing cells.
Affiliation: R.D. Berlin Center for Cell Analysis and Modeling and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030.