PKC-e Regulates Vesicle Delivery and Focal Exocytosis for Efficient IgG-mediated Phagocytosis

PKC-e is required for membrane addition during IgG-mediated phagocytosis; its role in this process is ill-defined. High resolution imaging revealed that PKC-e exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-e colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-e and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles are not delivered in macrophages from PKC-e null mice, or upon dissociation of the Golgi-associated pool of PKC-e, implicates Golgi-tethered PKC-e as a driver of Golgi-to-phagosome trafficking. Finally, we established that PKC-e's regulatory domain is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-e in focal exocytosis: its regulatory domain drives Golgi-derived vesicles to the phagosome while catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.

PKC-ε Regulates Vesicle Delivery and Focal Exocytosis for Efficient IgG-mediated Phagocytosis

PKC-ε is required for membrane addition during IgG-mediated phagocytosis; its role in this process is ill-defined. High resolution imaging revealed that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF-α and PKC-α colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF-α delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF-α+ vesicles are not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implicates Golgi-tethered PKC-ε as a driver of Golgi-to-phagosome trafficking. Finally, we established that PKC-ε's regulatory domain is sufficient for delivery of TNF-α+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis: its regulatory domain drives Golgi-derived vesicles to the phagosome while catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.

Asymmetric Cell Growth and Nucleoid Displacement inEscherichia coli

ABSTRACTSingle celled growth ofEscherichia coliis typically considered as an example symmetric division, based on the sizes of daughter cells and precision of center finding of the septum. Here, we investigate the symmetry of membrane addition in the mid-plane and DNA segregation using a video-microscopy approach. We find the membrane expansion dynamics to be asymmetric based on mid-cell photobleaching landmarks in FM4-64, used to stain the membrane. The apparent growth bias ofE. colidoes not correspond to the age of the pole. We find the membrane growth asymmetry is correlated to nucleoid displacement, consistent with ideas of coupling of cell growth and nucleoid positioning. The mobility of the actin-homolog MreB also correlates with membrane growth asymmetry, based on fluorescence recovery after photobleaching (FRAP) measurements of a YFP-fusion. These correlations suggest the small asymmetry of membrane addition observed could potentially drive nucleoid segregation and MreB mobility asymmetry inE. coli.IMPORTANCEAsymmetry in bacterial cell division is seen in it’s most simple form in the binomial event of protein segregation that results in ‘noise’ in equal segregation and depends on the protein copy number. In the case of specific proteins this can also affect growth, and result in differentiation. However, during the asexual division ofEscherichia colisingle cells are thought to grow symmetrically and divide equally. We find a slight but consistent asymmetry in growth based on quantitative morphometry of cell pole displacement in time-series of growingE. colicells. Increased cell wall extension in one half of the cell over the other appears to explain the asymmetry in the displacement of cell poles. Interestingly the growth asymmetry is not correlated to the age of the pole (old and new). This observed asymmetry appears to correlate with the asymmetry of nucleoid segregation, resulting in the nucleoids finding the midpoints of the respective daughter cells, and formation of the septum at the geometric centre of the elongated cell just prior to division. The mobility dynamics of the cytoskeletal protein MreB, which organizes the cell membrane, is are more dynamic where membrane growth is faster. Thus we propose a linkage between this observed growth asymmetry and that of MreB dynamics.

Fatty Acid Fueled Transmembrane Chloride Transport

We report an example of the use of fatty acids to drive chloride transport by creating a pH gradient across a vesicular lipid bilayer membrane. Addition of an unselective squaramide-based chloride transporter (which transports both H⁺and Cl^-) facilitates the transport of HCl from the vesicle (driven by the pH gradient) so creating a chloride gradient. Addition of further aliquots of fatty acid ‘fuel’ can initiate further transport of chloride out of the vesicle by re-establishing the pH gradient. This is an example of a prototypical chloride pumping system.

Fatty Acid Fueled Transmembrane Chloride Transport

We report an example of the use of fatty acids to drive chloride transport by creating a pH gradient across a vesicular lipid bilayer membrane. Addition of an unselective squaramide-based chloride transporter (which transports both H⁺and Cl^-) facilitates the transport of HCl from the vesicle (driven by the pH gradient) so creating a chloride gradient. Addition of further aliquots of fatty acid ‘fuel’ can initiate further transport of chloride out of the vesicle by re-establishing the pH gradient. This is an example of a prototypical chloride pumping system.

Fatty Acid Fueled Transmembrane Chloride Transport

We report an example of the use of fatty acids to drive chloride transport by creating a pH gradient across a vesicular lipid bilayer membrane. Addition of an unselective squaramide-based chloride transporter (which transports both H⁺and Cl^-) facilitates the transport of HCl from the vesicle (driven by the pH gradient) so creating a chloride gradient. Addition of further aliquots of fatty acid ‘fuel’ can initiate further transport of chloride out of the vesicle by re-establishing the pH gradient. This is an example of a prototypical chloride pumping system.

Fatty Acid-Fuelled Transmembrane Chloride Transport

We report an example of the use of fatty acids to drive chloride transport by creating a pH gradient across a vesicular lipid bilayer membrane. Addition of an unselective squaramide-based chloride transporter (which transports both H⁺and Cl^-) facilitates the transport of HCl from the vesicle (driven by the pH gradient) so creating a chloride gradient. Addition of further aliquots of fatty acid ‘fuel’ can initiate further transport of chloride out of the vesicle by re-establishing the pH gradient. This is an example of a prototypical chloride pumping system.

Dynamin-2 controls phagophore maturation

Autophagy involves rapid growth of phagophores through membrane addition. Newly added membranes are derived from other organelles through vesicles carrying the Atg9 protein. Membrane is delivered by fusing these vesicles with the phagophores. Atg9 is, nevertheless, not incorporated in autophagosomes. We now show that this protein is retrieved from phagophores by fission utilizing Dynamin-2 (Dnm2) as the membrane scission protein. Blocking Atg9 recycling by interfering with Dnm2 helps retain Atg9 in autophagosomes and degrades this protein by autophagy. Dnm2 colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1) when autophagy is induced, consistent with transient interactions during Atg9 retrieval. EndoB1 and Dnm2 also control the downstream fusion of phagophores to late endosomes, thus ensuring the completion of phagophores before proceeding to the next stage in the autophagy process. These data provide novel insights into the roles of membrane scission proteins during autophagy.