Phosphoinositides and engulfment

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108686/1/cmi12334.pd

The endoeytie activity of dendritic cells

[No abstract available

Calcium spikes in activated macrophages during Fcγ receptor‐mediated phagocytosis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142318/1/jlb0677.pd

Technical Advance: caspase‐1 activation and IL‐1β release correlate with the degree of lysosome damage, as illustrated by a novel imaging method to quantify phagolysosome damage

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142182/1/jlb0813.pd

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes

CXCL12‐induced macropinocytosis modulates two distinct pathways to activate mTORC1 in macrophages

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141686/1/jlb0683.pd

A Phosphatidylinositol-3-Kinase-Dependent Signal Transition Regulates ARF1 and ARF6 during Fcγ Receptor-Mediated Phagocytosis

Fcγ receptor (FcγR)–mediated phagocytosis of IgG-coated particles is regulated by 3′-phosphoinositides (3′PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcγR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcγR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome

Coordination of the Rab5 Cycle on Macropinosomes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88083/1/j.1600-0854.2011.01280.x.pd