activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Differ... more activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Different cell types express different subsets of isoforms. In tracheal epithelial cells, the activation of beta and epsilon isoforms by PMA was followed by their degradation. We sought to determine whether such activation-degradation patterns affected protrusion formation or turnover. Cells were treated by transcriptional knockdown (KD) and the prevalence of each protrusion class was analyzed by computerized morphometry. Latent factors for filopodia (#4) and nascent neurites (#7) were calculated based on geometric variables determined for known populations. At zero time, factor #7 was elevated by alpha KD. Control samples, into which a random KD sequence had been introduced, showed an increase in neurites throughout the PMA exposure. During the time course, all KD samples differed from controls at one or more times. Epsilon KD followed by PMA drastically downregulated the isoform by 15 h and decreased #7 values relative to control. Alpha KD was similar suggesting that the early KD effect (increasing #7) was reversed later on. Beta KD cells resembled control at 15 h but alpha KD at the 5-h time. Filopodia were eliminated by a 2-h PMA exposure regardless of the KD agent introduced. Control samples remained depressed, but epsilon and alpha KD samples partially recovered by 5 h. Alpha KD declined again dramatically by 15 h. The time courses suggest that the main effects were exerted by the actin-binding PKC epsilon, but alpha, which is not actin-binding, could have similar effects. The data suggest that competitive binding on RACKs may be occurring, complicating the picture of kinase regulation of adhesion and protrusive activities.
The surface of a living cell provides a platform for receptor signaling, protein sorting, transpo... more The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation re- quires the local control ofmembrane organization. Previouswork has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin.We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled viamembrane-associated actin- binding proteins to dynamic actin filaments andmyosinmotors.Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponentmembrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase- segregating domains. These results show how local membrane composition can be driven by active processes arising from actomy- osin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.
activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Differ... more activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Different cell types express different subsets of isoforms. In tracheal epithelial cells, the activation of beta and epsilon isoforms by PMA was followed by their degradation. We sought to determine whether such activation-degradation patterns affected protrusion formation or turnover. Cells were treated by transcriptional knockdown (KD) and the prevalence of each protrusion class was analyzed by computerized morphometry. Latent factors for filopodia (#4) and nascent neurites (#7) were calculated based on geometric variables determined for known populations. At zero time, factor #7 was elevated by alpha KD. Control samples, into which a random KD sequence had been introduced, showed an increase in neurites throughout the PMA exposure. During the time course, all KD samples differed from controls at one or more times. Epsilon KD followed by PMA drastically downregulated the isoform by 15 h and decreased #7 values relative to control. Alpha KD was similar suggesting that the early KD effect (increasing #7) was reversed later on. Beta KD cells resembled control at 15 h but alpha KD at the 5-h time. Filopodia were eliminated by a 2-h PMA exposure regardless of the KD agent introduced. Control samples remained depressed, but epsilon and alpha KD samples partially recovered by 5 h. Alpha KD declined again dramatically by 15 h. The time courses suggest that the main effects were exerted by the actin-binding PKC epsilon, but alpha, which is not actin-binding, could have similar effects. The data suggest that competitive binding on RACKs may be occurring, complicating the picture of kinase regulation of adhesion and protrusive activities.
The surface of a living cell provides a platform for receptor signaling, protein sorting, transpo... more The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation re- quires the local control ofmembrane organization. Previouswork has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin.We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled viamembrane-associated actin- binding proteins to dynamic actin filaments andmyosinmotors.Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponentmembrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase- segregating domains. These results show how local membrane composition can be driven by active processes arising from actomy- osin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.
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Papers by Kabir Husain