When wild-type (wt) tobacco (Nicotiana tabacum cv. Petit Havana SR1) cells are grown under macronutrient (P or N) limitation, they induce large amounts of alternative oxidase (AOX), which constitutes a non-energy-conserving branch of the respiratory electron transport chain. To investigate the significance of AOX induction, wt cells were compared with transgenic (AS8) cells lacking AOX. Under nutrient limitation, growth of wt cell cultures was dramatically reduced and carbon use efficiency (g cell dry weight gain g(-1) sugar consumed) decreased by 42-63%. However, the growth of AS8 was only moderately reduced by the nutrient deficiencies and carbon use efficiency values remained the same as under nutrient-sufficient conditions. As a result, the nutrient limitations more severely compromised the tissue nutrient status (P or N) of AS8 than wt cells. Northern analyses and a comparison of the mitochondrial protein profiles of wt and AS8 cells indicated that the lack of AOX in AS8 under P limitation was associated with increased levels of proteins commonly associated with oxidative stress and/or stress injury. Also, the level of electron transport chain components was consistently reduced in AS8 while tricarboxylic acid cycle enzymes did not show a universal trend in abundance in comparison to the wt. Alternatively, the lack of AOX in AS8 cells under N limitation resulted in enhanced carbohydrate accumulation. It is concluded that AOX respiration provides an important general mechanism by which plant cells can modulate their growth in response to nutrient availability and that AOX also has nutrient-specific roles in maintaining cellular redox and carbon balance.

Phagocytosis is a complex sequence of events involving coordinated remodeling of the plasma membrane with the underlying cytoskeleton. Although the role of the actin cytoskeleton is becoming increasingly elucidated, the role of microtubules (MTs) remains poorly understood. Here, we examine the role of MTs during FcgammaR-mediated phagocytosis in RAW264.7 mouse macrophages. We observe that MTs extend into the phagosomal cups. The MT-depolymerizing agents, colchicine and nocodazole, cause a sizeable reduction in phagocytosis of large particles in RAW264.7 cells. Phagocytosis in primed macrophages is unaffected by MT-depolymerizing agents. However, activation of macrophages coincides with an increased population of drug-stable MTs, which persist in functional phagocytic cups. Scanning electron microscopy analysis of unprimed macrophages reveals that pseudopod formation is reduced markedly following colchicine treatment, which is not a consequence of cell rounding. MT depolymerization in these cells does not affect particle binding, Syk, or Grb2-associated binder 2 recruitment or phosphotyrosine accumulation at the site of phagocytosis. Ras activation also proceeds normally in macrophages treated with colchicine. However, MT disruption causes a decrease in accumulation of AKT-pleckstrin homology-green fluorescent protein, a probe that binds to PI-3K products at the sites of particle binding. A corresponding decline in activated AKT is observed in colchicine-treated cells using immunoblotting with a phospho-specific-AKT (ser473) antibody. Furthermore, the translocation of the p85alpha regulatory subunit of PI-3K is reduced at the phagocytic cup in colchicine-treated cells. These findings suggest that MTs regulate the recruitment and localized activity of PI-3K during pseudopod formation.

Macrophages can suppress Chlamydia replication by targeting the bacteria to degradative organelles such as lysosomes.

Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 m are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by Fc␥R ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages. INTRODUCTIONPhagocytosis is a specialized mechanism for cells of the innate immune system to clear pathogens and dying cells from the body. Phagocytosis is initiated at the site of particle/pathogen attachment, creating a polarized region of activity within the macrophage. This process is a rapid, highly orchestrated event that recruits a multitude of signaling proteins, mobilizes organelles, and causes dramatic cytoskeletal rearrangements. Phagocytosis is initiated by ligation of Fc␥ receptors to IgG-opsonins on the target cell. Engaged Fc␥Rs cluster at the site of particle contact, which in turn recruits Src, Syk, and PI3K (Greenberg and Grinstein, 2002). Local activation of cdc42 and rac1 initiates actin remodeling that coincides with the growth of membrane pseudopods (Greenberg and Grinstein, 2002). Within minutes, the particles are engulfed by the pseudopods into the cytoplasm as a membrane-bound phagosome. Particle contents within the phagosome are then degraded by fusion with the macrophage endocytic machinery. Phagolysosome formation is concomitant with retrograde translocation of the phagosome along the microtubule (MT) cytoskeleton (Blocker et al., 1997;Harrison and Grinstein, 2002;Harrison et al., 2003). Over the next several hours, particulate antigens from the phagosome will bind to specialized antigen-presentation proteins that will become displayed on the cell surface to initiate the adaptive immune response. Exogenous antigens bind to Major Histocompatibility Complex II (MHCII) proteins, which are largely delivered to phagosomes from Golgi-derived endocytic organelles (Ramachandra and Harding, 2000). Macrophages are also capable of cross-presenting antigens that become complexed with MHC I molecules in the endoplasmic reticulum (ER), before surface presentation (G...