Intracellular delivery and deep tissue penetration of nucleoside triphosphates using photocleavable covalently bound dendritic polycations.

Nucleoside triphosphates (NTPs) are essential in various biological processes. Cellular or even organismal controlled delivery of NTPs would be highly desirable, yet in cellulo and in vivo applications are hampered owing to their negative charge leading to cell impermeability. NTP transporters or NTP prodrugs have been developed, but a spatial and temporal control of the release of the investigated molecules remains challenging with these strategies. Herein, we describe a general approach to enable intracellular delivery of NTPs using covalently bound dendritic polycations, which are derived from PAMAM dendrons and their guanidinium derivatives. By design, these modifications are fully removable through attachment on a photocage, ready to deliver the native NTP upon irradiation enabling spatiotemporal control over nucleotide release. We study the intracellular distribution of the compounds depending on the linker and dendron generation as well as side chain modifications. Importantly, as the polycation is bound covalently, these molecules can also penetrate deeply into the tissue of living organisms, such as zebrafish.

Spatiotemporal Regulation of FMNL2 by N-Terminal Myristoylation and C-Terminal Phosphorylation Drives Rapid Filopodia Formation.

The actin nucleating and polymerizing formin-like 2 (FMNL2) is upregulated in several cancers and has been shown to play important roles in cell migration, invasion, cell-cell adhesion and filopodia formation. Here, using structured illumination microscopy we show that FMNL2 promotes rapid and highly dynamic filopodia formation in epithelial cells while remaining on the tip of the growing filopodia. This filopodia tip localization depends fully on its N-terminal myristoylation. We further show that FMNL2-dependent filopodia formation requires its serine 1072 phosphorylation within the diaphanous-autoregulatory domain (DAD) by protein kinase C (PKC) α. Consistent with this, filopodia formation depends on PKC activity and PKCα localizes to the base of growing filopodia. Thus, a PKCα-FMNL2 signaling module spatiotemporally controls dynamic filopodia formation.

Vesicle-Associated Actin Assembly by Formins Promotes TGFß-Induced ANGPTL4 Trafficking, Secretion and Cell Invasion.

Vesicle trafficking has emerged as an important process driving tumor progression through various mechanisms. Transforming growth factor beta (TGFß)-mediated secretion of Angiopoietin-like 4 (ANGPTL4) is important for cancer development. Here, Formin-like 2 (FMNL2) is identified to be necessary for ANGPTL4 trafficking and secretion in response to TGFß. Protein kinase C (PKC)-dependent phosphorylation of FMNL2 downstream of TGFß stimulation is required for cancer cell invasion as well as ANGPTL4 vesicle trafficking and secretion. Moreover, using super resolution microscopy, ANGPTL4 trafficking is actin-dependent with FMNL2 directly polymerizing actin at ANGPTL4-containing vesicles, which are associated with Rab8a and myosin Vb. This work uncovers a formin-controlled mechanism that transiently polymerizes actin directly at intracellular vesicles to facilitate their mobility. This mechanism may be important for the regulation of cancer cell metastasis and tumor progression.