Actin-independent mechanisms of targeting formin mDIA2 to the plasma membrane

The actin cytoskeleton is absolutely essential for metazoan life at all stages. In cells, actin polymerizes into filaments that assemble into unique structures, which perform essential functions. Assembly of these structures depends on recruitment of actinregulatory proteins to specific cellular locations, a complex process known as subcellular targeting. Formins are important actin-regulatory proteins that assemble unbranched actin structures. The mechanisms by which formins are targeted to their subcellular sites are poorly understood, but are known to rely in part on binding Rho GTPases. Formin mDia2 plays various cellular roles and promotes cellular motility by elongating actin filaments in lamellipodia and filopodia. Localization of mDia2 to these edge protrusions depends on interactions of mDia2 with actin filaments and the plasma membrane. Whereas the mechanism of binding actin filaments by mDia2 is well-established, how mDia2 binds the plasma membrane is poorly understood. Particularly, domains that mediate binding to the plasma membrane are unknown. In addition, it is unclear what GTPases target mDia2 to the plasma membrane. We investigated the mechanism of targeting of mDia2 to the plasma membrane and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing FH1-FH2 module was strongly localized on the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the Diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N-terminus required the presence of both G and DID. These results suggest that the entire N-terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif

Actin-independent mechanisms of targeting formin mDIA2 to the plasma membrane

The actin cytoskeleton is absolutely essential for metazoan life at all stages. In cells, actin polymerizes into filaments that assemble into unique structures, which perform essential functions. Assembly of these structures depends on recruitment of actinregulatory proteins to specific cellular locations, a complex process known as subcellular targeting. Formins are important actin-regulatory proteins that assemble unbranched actin structures. The mechanisms by which formins are targeted to their subcellular sites are poorly understood, but are known to rely in part on binding Rho GTPases. Formin mDia2 plays various cellular roles and promotes cellular motility by elongating actin filaments in lamellipodia and filopodia. Localization of mDia2 to these edge protrusions depends on interactions of mDia2 with actin filaments and the plasma membrane. Whereas the mechanism of binding actin filaments by mDia2 is well-established, how mDia2 binds the plasma membrane is poorly understood. Particularly, domains that mediate binding to the plasma membrane are unknown. In addition, it is unclear what GTPases target mDia2 to the plasma membrane. We investigated the mechanism of targeting of mDia2 to the plasma membrane and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing FH1-FH2 module was strongly localized on the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the Diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N-terminus required the presence of both G and DID. These results suggest that the entire N-terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif

Wall shear stress from a single almost spherical and a Taylor bubbles in laminar upward tube flow

An experimental electrodiffusional technique with eight double probes is used to detect perturbation the wall shear stress caused by a single bubble in laminar upward tube flow. A small almost spherical and different length Taylor bubbles are considered. The shear stress perturbations by bubbles have a complex structure. It is possible to define three components of perturbation caused by a small bubble. The perturbation by Taylor bubble contains only two components due to the main flow symmetry around the bubble. An unexpectedly long shear stress pulsations zone is registered behind the bubbles

Wall shear stress from a single almost spherical and a Taylor bubbles in laminar upward tube flow

An experimental electrodiffusional technique with eight double probes is used to detect perturbation the wall shear stress caused by a single bubble in laminar upward tube flow. A small almost spherical and different length Taylor bubbles are considered. The shear stress perturbations by bubbles have a complex structure. It is possible to define three components of perturbation caused by a small bubble. The perturbation by Taylor bubble contains only two components due to the main flow symmetry around the bubble. An unexpectedly long shear stress pulsations zone is registered behind the bubbles