Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

Abstract

Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor Fc␥RIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing Fc␥RIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of Fc␥RIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome.

Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling

“…Using previously established quantitative microfluorometry and high-speed fluorescence microscopy methods, we have studied the Ca 2ϩ signaling events associated with spontaneous neutrophil motility. Using an efficient optical system and photodetector capable of sensing single atoms, we have been able to achieve electronic shutter speeds of ϳ0.1 s, which allows us to follow the intracellular trafficking of intracellular signals (10,12,13). Our recent study (10) and the experiments described above indicate that the plasma membrane component of this signal originates at the lipid raft-like lamellipodium.…”

“…We have recently developed high-speed optical microscopy, which allows us to detect chemical signals within small cells with unprecedented detail (10,12,13). A previous high-speed microscopy study from this laboratory showed that each repetitive Ca 2ϩ spike corresponded to a Ca 2ϩ wave traveling around the neutrophil's periphery (10).…”

“…To increase light collection efficiency, the microscope's bottom port was fiber-optically coupled to the input side of an Acton-150 (Acton Instruments, Acton, MA) imaging spectrophotometer. The exit side was connected to a liquid N 2 -cooled intensifier attached to a Peltier-cooled I-MAX-512 camera (approximately Ϫ20°C) (Princeton Instruments) (12,13). …”

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…The studies reported above provide some insight into the nature of Ca 2ϩ signaling associated with cell motility. On the basis of these and earlier (29,35) studies, we suggest that the initial Ca 2ϩ "flash" or "bump" is associated with the endoplasmic reticulum or a component of the endoplasmic reticulum closely associated with the plasma membrane. Alternatively, the TRP channel could be responding to cytoskeletal interactions or changes in membrane stress (63,64).…”

“…Our studies provide new insights into tumor cell migration and unprecedented detail concerning the cellular effects of Ca 2ϩ -active reagents on tumor cells. As we have emphasized (29,35,60), intracellular Ca 2ϩ waves, which are the Ca 2ϩ signal, can be very heterogeneous in their physical A final concentration of 25 g/ml fibronectin was added to the bottom chamber medium as a chemoattractant. After a 22-h incubation, transmigrated cells on the bottom side of the inserts were fixed, stained, and counted.…”