Mitochondria are known as the powerhouses of eukaryotic cells; however, they perform many other functions besides oxidative phosphorylation, including Ca²⁺ homeostasis, lipid metabolism, antiviral response, and apoptosis. Although other hypotheses exist, mitochondria are generally thought as descendants of an α-proteobacteria that adapted to the intracellular environment within an Asgard archaebacteria, which have been studied for decades as an organelle subdued by the eukaryotic cell. Nevertheless, several early electron microscopy observations hinted that some mitochondria establish specific interactions with certain plasma membrane (PM) domains in mammalian cells. Furthermore, recent findings have documented the direct physical and functional interaction of mitochondria and the PM, the organization of distinct complexes, and their communication through vesicular means. In yeast, some molecular players mediating this interaction have been elucidated, but only a few works have studied this interaction in mammalian cells. In addition, mitochondria can be translocated among cells through tunneling nanotubes or by other mechanisms, and free, intact, functional mitochondria have been reported in the blood plasma. Together, these findings challenge the conception of mitochondria as organelles subdued by the eukaryotic cell. This review discusses the evidence of the mitochondria interaction with the PM that has been long disregarded despite its importance in cell function, pathogenesis, and evolution. It also proposes a scheme of mitochondria–PM interactions with the intent to promote research and knowledge of this emerging pathway that promises to shift the current paradigms of cell biology.

Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa²⁺ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa²⁺ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca²⁺ depletion, indicating flux-independent NMDAR function. In contrast, the IP₃ receptor (IP₃R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa²⁺ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa²⁺ increase.

The glutamate (Glu) N-methyl-d-aspartate (NMDA) receptor (NMDAR) plays a critical role in synaptic communication given mainly by its ionotropic function that permeates Ca²⁺. This in turn activates calmodulin that triggers CaMKII, MAPK, CREB, and PI3K pathways, among others. However, NMDAR signaling is more complex. In the last two decades several groups have shown that the NMDAR also elicits flux-independent signaling (f-iNMDARs). It has been demonstrated that agonist (Glu or NMDA) or co-agonist (Glycine or d-Serine) bindings initiate intracellular events, including conformational changes, exchange of molecular interactions, release of second messengers, and signal transduction, that result in different cellular events including endocytosis, LTD, cell death, and neuroprotection, among others. Interestingly, f-iNMDARs has also been observed in pathological conditions and non-neuronal cells. Here, the molecular and cellular events elicited by these flux-independent actions (non-canonical or metabotropic-like) of the NMDAR are reviewed. Considering the NMDAR complexity, it is possible that these flux-independent events have a more relevant role in intracellular signaling that has been disregarded for decades. Moreover, considering the wide expression and function of the NMDAR in non-neuronal cells and other tissues beyond the nervous system and some evolutionary traits, f-iNMDARs calls for a reconsideration of how we understand the biology of this complex receptor.

Glutamate N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca ²⁺ channel. Nonetheless, flux‐independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) acting through the NMDAR elicits flux‐independent Ca ²⁺ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca ²⁺ release is due to pH6 sensing by NMDAR, whereas mΔΨ depletion requires both: pH6 and flux‐dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non‐random distribution relative to the ER and mitochondria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria‐ER, and ‐nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM‐mitochondrial bridges (PM‐m‐br). These and earlier data strongly suggest PM‐mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM‐4‐64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca ²⁺ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demonstrate that NMDAR has concomitant flux‐independent and flux‐dependent actions in cultured astrocytes.

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Ralpha, -beta, and -gamma(c)). IL-2Rbeta is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rbeta is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37beta ic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rbeta decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37Beta ic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rbeta mimicking 37beta ic increases their proliferation. These data indicate that IL-2Rbeta is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation.

The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may participate. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecules extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases, mechanism known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecules intracellular domain (ICD) that carries out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i- CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments.