Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to "swarms" and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP3R-mediated Ca2+ release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP3R Ca2+ signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca2+ uptake. Mitochondrial uptake of InsP3R released Ca2+ is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca2+ transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates pro-survival macroautophagy. Thus, constitutive InsP3R Ca2+ release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Increasing evidence suggests that amyloid peptides associated with a variety of degenerative diseases induce neurotoxicity in their intermediate oligomeric state, rather than as monomers or fibrils. To test this hypothesis and investigate the possible involvement of Ca 2؉ signaling disruptions in amyloid-induced cytotoxicity, we made homogeneous preparations of diseaserelated amyloids (A␤, prion, islet amyloid polypeptide, polyglutamine, and lysozyme) in various aggregation states and tested their actions on fluo-3-loaded SH-SY5Y cells. Application of oligomeric forms of all amyloids tested (0.6 -6 g ml ؊1 ) rapidly (ϳ5 s) elevated intracellular Ca 2؉ , whereas equivalent amounts of monomers and fibrils did not. Ca 2؉ signals evoked by A␤42 oligomers persisted after depletion of intracellular Ca 2؉ stores, and small signals remained in Ca 2؉ -free medium, indicating contributions from both extracellular and intracellular Ca 2؉ sources. The increased membrane permeability to Ca 2؉ cannot be attributed to activation of endogenous Ca 2؉ channels, because responses were unaffected by the potent Ca 2؉ -channel blocker cobalt (20 m). Instead, observations that A␤42 and other oligomers caused rapid cellular leakage of anionic fluorescent dyes point to a generalized increase in membrane permeability. The resulting unregulated flux of ions and molecules may provide a common mechanism for oligomer-mediated toxicity in many amyloidogenic diseases, with dysregulation of Ca 2؉ ions playing a crucial role because of their strong trans-membrane concentration gradient and involvement in cell dysfunction and death.Alzheimer disease (AD) 1 is characterized by the appearance in the brain of plaques, containing extracellular deposits of amyloid ␤-peptide (A␤) that result from altered proteolytic processing of amyloid precursor protein, together with intracellular neurofibrillary tangles containing misfolded tau (1).Brain regions with plaques and tangles exhibit reduced numbers of synapses, and neurites associated with plaques and tangles are often damaged, suggesting a pivotal role for A␤ in the neuropathology of AD (2-5). Moreover, numerous other neurodegenerative disorders (including Huntington, Parkinson, and prion diseases) are also associated with the formation and accumulation of amyloid fibrils in specific brain areas (6, 7). These commonalities suggest a general mechanism of action for the more than 100 human amyloid-related diseases, whereby normally soluble peptides and proteins undergo aberrant folding (8).Aggregation of A␤ proceeds through several conformational states, including dimers, spherical oligomers composed of 10 -24 monomers, and strings of oligomers (protofibrils), before finally assuming an insoluble fibrillar conformation (9). The initial formulation of the "amyloid hypothesis" of AD specifically implicated fibrillar amyloid deposits (10). However, more recent evidence suggests that soluble oligomers may be the principal neurotoxic agent (11-15). Soluble A␤ oligomers are found in the cerebrospinal fluid of ...

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo–labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11–12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events.

Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone–T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 μm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell–T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell–dependent antibody responses.