João F.V. Barroso

Tunneling nanotubes: A new route for the exchange of components between animal cells

Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell‐to‐cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.

Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells

Organelle exchange between cells via tunneling nanotubes (TNTs) is a recently described form of intercellular communication. Here, we show that the selective elimination of filopodia from PC12 cells by 350 nM cytochalasin B (CytoB) blocks TNT formation but has only a weak effect on the stability of existing TNTs. Under these conditions the intercellular organelle transfer was strongly reduced, whereas endocytosis and phagocytosis were not affected. Furthermore, the transfer of organelles significantly correlated with the presence of a TNT‐bridge. Thus, our data support that in PC12 cells filopodia‐like protrusions are the principal precursors of TNTs and CytoB provides a valuable tool to selectively interfere with TNT‐mediated cell‐to‐cell communication.

Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells ☆

Tunneling nanotube (TNT)-like structures are intercellular membranous bridges that mediate the transfer of various cellular components including endocytic organelles. To gain further insight into the magnitude and mechanism of organelle transfer, we performed quantitative studies on the exchange of fluorescently labeled endocytic structures between normal rat kidney (NRK) cells. This revealed a linear increase in both the number of cells receiving organelles and the amount of transferred organelles per cell over time. The intercellular transfer of organelles was unidirectional, independent of extracellular diffusion, and sensitive to shearing force. In addition, during a block of endocytosis, a significant amount of transfer sustained. Fluorescence microscopy revealed TNT-like bridges between NRK cells containing F-actin but no microtubules. Depolymerization of F-actin led to the disappearance of TNT and a strong inhibition of organelle exchange. Partial ATP depletion did not affect the number of TNT but strongly reduced organelle transfer. Interestingly, the myosin II specific inhibitor S-(-)-blebbistatin strongly induced both organelle transfer and the number of TNT, while the general myosin inhibitor 2,3-butanedione monoxime induced the number of TNT but significantly inhibited transfer. Taken together, our data indicate a frequent and continuous exchange of endocytic organelles between cells via TNT by an actomyosin-dependent mechanism.

Probing the role of crystallographically defined/predicted hinge-bending regions in the substrate-induced global conformational transition and catalytic activation of human phenylalanine hydroxylase by single-site mutagenesis.

Phenylalanine hydroxylase (PAH) is generally considered to undergo a large and reversible conformational transition upon l-Phe binding, which is closely linked to the substrate-induced catalytic activation of this hysteretic enzyme. Recently, several crystallographically solvent-exposed hinge-bending regions including residues 31-34, 111-117, 218-226, and 425-429 have been defined/predicted to be involved in the intra-protomer propagation of the substrate-triggered molecular motions generated at the active site. On this basis, single-site mutagenesis of key residues in these regions of the human PAH tetramer was performed in the present study, and their functional impact was measured by steady-state kinetics and the global conformational transition as assessed by surface plasmon resonance and intrinsic tryptophan fluorescence spectroscopy. A strong correlation (r2 = 0.93-0.96) was observed between the l-Phe-induced global conformational transition and Vmax values for wild-type human PAH and the mutant forms K113P, N223D, N426D, and N32D, in contrast to the substitution T427P, which resulted in a tetrameric form with no kinetic cooperativity. Furthermore, the flexible intra-domain linker region (residues 31-34) seems to be involved in a more local conformational change, and the biochemical/biophysical properties of the G33A/G33V mutant forms support a key function of this residue in the positioning of the autoregulatory sequence (residues 1-30) and thus in the regulation of the solvent and substrate access to the active site. The mutant forms revealed a variably reduced global conformational stability compared with wild-type human PAH, as measured by thermal denaturation and limited proteolysis.

Distinct roles of myosin Va in membrane remodeling and exocytosis of secretory granules.

Hormone‐ and neuropeptide‐containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans‐ Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin‐coated vesicles. The resulting mature SGs undergo Ca2+‐dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F‐actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP‐inhibited binding of SGs to F‐actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant‐negative myosin Va‐tail or shRNA‐based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary, our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.

Transient Up‐Regulation of Liver Mitochondrial Thymidine Kinase Activity in Proliferating Mitochondria

Administration of the fatty acid analogue tetradecylthioacetic acid (TTA) to rodents up‐regulates peroxisomal and mitochondrial lipid‐metabolizing enzymes and induces a proliferation of these organelles in hepatocytes. We show here that male NMRI mice fed a diet containing 0.3% (w/w) TTA revealed a transient two‐fold increase in the incorporation of [3