Plasma Membrane Ca2+ ATPase Activity Enables Sustained Store-operated Ca2+ Entry in the Absence of a Bulk Cytosolic Ca2+ Rise.

In many cell types, the rise in cytosolic Ca2+ due to opening of Ca2+ release-activated Ca2+ (CRAC) channels drives a plethora of responses, including secretion, motility, energy production, and gene expression. The amplitude and time course of the cytosolic Ca2+ rise is shaped by the rates of Ca2+ entry into and removal from the cytosol. However, an extended bulk Ca2+ rise is toxic to cells. Here, we show that the plasma membrane Ca2+ ATPase (PMCA) pump plays a major role in preventing a prolonged cytosolic Ca2+ signal following CRAC channel activation. Ca2+ entry through CRAC channels leads to a sustained sub-plasmalemmal Ca2+ rise but bulk Ca2+ is kept low by the activity of PMCA4b. Despite the low cytosolic Ca2+, membrane permeability to Ca2+ is still elevated and Ca2+ continues to enter through CRAC channels. Ca2+-dependent NFAT activation, driven by Ca2+ nanodomains near the open channels, is maintained despite the return of bulk Ca2+ to near pre-stimulation levels. Our data reveal a central role for PMCA4b in determining the pattern of a functional Ca2+ signal and in sharpening local Ca2+ gradients near CRAC channels, whilst protecting cells from a toxic Ca2+ overload.

AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels.

To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Signaling through Ca2+ Microdomains from Store-Operated CRAC Channels.

Calcium (Ca2+) ion microdomains are subcellular regions of high Ca2+ concentration that develop rapidly near open Ca2+ channels in the plasma membrane or internal stores and generate local regions of high Ca2+ concentration. These microdomains are remarkably versatile in that they activate a range of responses that differ enormously in both their temporal and spatial profile. In this review, we describe how Ca2+ microdomains generated by store-operated calcium channels, a widespread and conserved Ca2+ entry pathway, stimulate different signaling pathways, and how the spatial extent of a Ca2+ microdomain can be influenced by Ca2+ ATPase pumps.

Kinesin-dependent mechanism for controlling triglyceride secretion from the liver.

Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER-LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs.

Reconstitution of microtubule-dependent organelle transport.

Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles.

Teamwork in microtubule motors.

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.

Quantitative optical trapping on single organelles in cell extract.

We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.

Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes.

Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.