Requirement of Dynactin p150Glued Subunit for the Functional Integrity of the Keratinocyte Microparasol

Abstract

The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.

Mimicking Melanosomes: Polydopamine Nanoparticles as Artificial Microparasols

“…10,29 Second, synthetic fluorescent microspheres had been used to establish the role of the dynactin p150Glued subunit as a required part of the cellular machinery for keratinocytes in which the knockout showed a lack of microparasol formation. 30 To test our hypothesis, we first synthesized spherical MelNPs by spontaneous oxidization of dopamine under alkaline conditions, introducing aqueous ammonium hydroxide to an aqueous solution of monomers (Figure 1). 31 The resulting spherical MelNPs showed a narrow size distribution around 200 nm, observed by transmission electron microscopy (TEM) (Figure 1a), scanning electron microscopy (SEM) (Figure 1b) and dynamic light scattering (DLS) (Figure S1).…”

“…This suggests that the transport process may be dependent on particle type, and that polydopamine nanoparticle surface chemistry plays a role in governing cellular distribution patterns. 30 …”

Melanin’s Journey from Melanocytes to Keratinocytes: Uncovering the Molecular Mechanisms of Melanin Transfer and Processing

“…The same study also reported that treatment with anti-sense DNA for cytoplasmic dynein heavy chain leads to a significant dispersion of perinuclear melanin in human keratinocytes. Subsequent studies demonstrated that the silencing of the dynactin p150 Glued subunit impairs the perinuclear clustering of microspheres in human keratinocytes [122], which suggests that melanin polar-ization is dependent on the cytoplasmatic dynein-dynactin motor complex (Figure 5). These results were further corroborated by recent studies, which reported a significant impairment in melanin polarization upon disruption of the actin cytoskeleton and the microtubule network [123].…”

Biology of melanocytes in mammals

“…Subsequently, the melanocores are internalized by keratinocytes through phagocytosis, which relies on the activation of protease-activated receptor-2 (PAR-2) (Wu and Hammer, 2014;Tadokoro and Takahashi, 2017;Moreiras et al, 2022). Following the transfer of melanin to keratinocytes, the cytoplasmic intermediate protein chains of dynein (Dync1i1) link the entire motor complex and the p150 Glued (DCTN1) subunit of the dynactin complex, which regulates the localization of perinuclear melanin (Byers et al, 2003;Byers et al, 2007). The calcium-dependent G protein-coupled and Akt signaling pathways upregulate the expression of DCTN1 in keratinocytes after exposure to UVA radiation.…”