Class I phosphoinositide 3-kinase (PI3K) is a lipid kinase playing key roles in many signaling pathways regulating cell survival and growth. Besides its important role in signal transduction, PI3K is also involved in actin and membrane reorganization such as protrusion, adhesion, phagocytosis and macropinocytosis. Receptor-mediated endocytosis is initiated by plasma membrane reorganization creating buds that then mature to small vesicles. Whereas most of endocytic mechanisms involve actin polymerization, PI3K requirement has not been clearly investigated. Our study identifies class I PI3K as a key player in clathrin-independent endocytosis of the interleukin 2 receptor (IL-2R) in contrast to the clathrin-dependent entry of transferrin (Tf). IL-2R is a cytokine receptor, inducing several signaling cascades such as PI3K, that are essential for the immune response. We have shown previously that IL-2R can be internalized with or without IL-2 and this process requires dynamin, actin and their regulators cortactin, N-WASP, Rac1 and the kinases Pak. Our recent work reveals that PI3K regulates Rac1 during IL-2R uptake in two ways: via its catalytic activity (p110) and via its regulatory factor (p85). Indeed, the catalytic activity of PI3K is required for both constitutive and IL-2 induced uptake of cytokine receptors, in lymphocytes as well as in epithelial cells. Interestingly, Vav2, a Rac1 GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved and recruited during IL-2R uptake. The second action of PI3K is via its regulatory subunit, p85, which binds activated Rac1 and IL-2R; this interaction being enhanced upon IL-2 treatment. Thus, PI3K regulates both the activation of Rac1 and its recruitment during IL-2R endocytosis. Finally, our results identify a link between cytokine receptors signaling and clathrin-independent endocytosis.
cytokine; receptor-mediated endocytosis; Rac1; signal transduction; PI3K; plasma membrane
Microglia are the primary immune cells in the brain. Under pathological conditions, they become activated and participate in scavenging, inflammation and tissue repair in response to brain injury. While the function and underlying mechanism of activated microglia have been intensively studied in the past decades, physiological functions of resting microglia remain largely underestimated. In our recent work, by simultaneously monitoring both the motility of resting microglial processes and the activity of surrounding neurons in intact zebrafish optic tectum, we examined the interaction between resting microglia and neurons. Local increase in neuronal activity attracts resting microglial processes and drives them to contact neurons with high levels of activity. This process is mediated by neuronal release of “find-me” signals such as ATP via pannexin-1 hemichannels and requires small Rho GTPase Rac in microglia. Reciprocally, the microglia-neuron contact reduces both the spontaneous and visually evoked activities of contacted neurons. We here summarize and explain the key results in the context of our previous work.
resting microglia; neuronal activity; microglia-neuron contact; pannexin-1 hemichannel; optic tectum; glutamate uncaging; in vivo two-photon imaging; calcium imaging; in vivo whole-cell recording; zebrafish
Maintaining the dynamic proteome of a living cell in the face of an ever-changing environment depends on a fine-tuned balance of protein synthesis and protein degradation. Molecular chaperones exert key functions during protein homeostasis (proteostasis). They associate with nonnative client proteins following synthesis or damage and facilitate client sorting and folding. When client proteins are terminally misfolded, chaperones cooperate with protein degradation systems to dispose of such clients. This dual proteostasis activity of chaperones is essential for maintaining cell function under normal growth conditions and becomes even more important under stress conditions such as heat and oxidative stress. The recent identification of chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway highlights the critical role of molecular chaperones in mechanically strained cells and tissues. The CASA complex, assembled by the cochaperone BAG3, coordinates protein degradation and protein synthesis in response to mechanical force. Here we describe the composition and function of this chaperone complex in mammals and discuss its relevance for tissue homeostasis and the regulation of cell adhesion, migration and proliferation. We provide a unifying concept for the function of BAG3, which integrates its involvement in muscle maintenance, tumor formation and virus infection.
BAG3; CASA; chaperone; ubiquitin; autophagy; mechanotransduction; Hsp70; WW domain
Aberrant regulation of glycogen synthase kinase-3 (GSK-3) is implicated in Alzheimer’s disease (AD), but the mechanisms involved remain elusive. Our recent study shows that GSK-3 impairs lysosomal acidification and that inhibition of GSK-3 re-acidified lysosomes in brains of AD mice. This effect was accompanied by reductions in β-amyloid pathology and amelioration of cognitive deficits. Presenilin-1 (PS1) is an essential factor in lysosomal acidification. To determine whether the inhibition of GSK-3 restores lysosomal malfunction caused by dysfunctional PS1, we treated MEF cells deficient in presenilin proteins (MEF-PS1/2−/−) with a selective substrate competitive GSK-3 inhibitor, L803-mts. L803-mts enhanced the acidic lysosomal pool in MEF-PS1/2−/− cells and increased levels of activated cathepsin D in the lysosomes. We conclude that GSK-3 and PS1 operate via similar mechanisms to disrupt lysosomal acidification. Importantly, these data indicate that GSK-3 inhibitors have potential in treatment of conditions associated with defective PS1.
Alzheimer’s disease; Aβ pathology; GSK-3; lysosome; presenilin-1
The Reed-Sternberg (RS) cell is the driving force behind Hodgkin lymphoma (HL), a unique malignancy in which the rare RS cell creates an inflammatory microenvironment that recruits a reactive tumor infiltrate. Well-known oncogenic factors such as nuclear factor kappa B (NFκB) signaling and Epstein-Barr virus infection are linked to HL pathogenesis but do not adequately explain the RS cell’s key pathologic features of multi-nucleation, abnormalities of centrosome function and number and aneuploidy. Chromosomal instability is also considered a key pathway in the origin of the RS cell, though the molecular mechanisms have largely been a “black box.” We demonstrated that the midbody kelch domain protein KLHDC8B protects against mitotic errors, centrosomal amplification and chromosomal instability. Here we discuss how the new findings linking KLHDC8B to mitotic integrity and faithful chromosomal segregation are providing mechanistic explanations for the origin of the RS cell and the molecular pathogenesis of chromosomal instability in HL.
Hodgkin lymphoma; Reed-Sternberg cell; aneuploidy; micronuclei; chromothripsis; kelch proteins; mitosis
Processing of sensory information from both sides of the body requires coordination of sensory input between the two hemispheres. This coordination is achieved by transcallosal (interhemispheric) fibers that course though the upper cortical layers. In a recent study by Palmer et al. (2012), we investigated the role of this interhemispheric input on the dendritic and somatic activity of cortical pyramidal neurons. This study showed that interhemispheric input evokes GABAB-mediated inhibition in the distal dendrites of layer 5 pyramidal neurons, decreasing the action potential output when paired with contralateral sensory stimulation. In contrast, layer 2/3 pyramidal neurons were not inhibited by interhemispheric input, possibly due to transcallosal fibers evoking more excitation in these neurons than layer 5 neurons. These results highlight both the precise nature of the microcircuitry of interhemispheric inhibition and how the balance between excitation and inhibition is different in the different layers of the cortex. Identifying the cellular and molecular elements involved in interhemipsheric inhibition is crucial not only for understanding higher brain function and but also dysfunction in the diseased brain.
interhemispheric inhibition; GABAB; corpus callosum; dendrite; cortical pyramidal neurons
Release and reception of extracellular ATP by leukocytes plays a critical role in immune responses to infection, injury and cardiovascular disease. Leukocytes of both the innate, adaptive immune and central nervous system express a repertoire of cell surface receptors for ATP (P2X and P2Y receptors) and its metabolites. ATP acts as a damage-associated molecule pattern (DAMP) released by injured or dying cells. Detection of released ATP by neighboring leukocytes initiates inflammation and wound healing. However, recent evidence from our group and others suggests ATP release by leukocytes themselves serves to regulate homeostatic mechanisms and coordinate responses to external pro-inflammatory cues. Examples include the homeostatic control of intracellular calcium and regulation of migratory guidance during chemotactic response to external cues. Though there has been some progress in elucidating ATP release mechanisms of some mammalian cells types, release conduits and coupling signal transduction machinery remain larger elusive for leukocytes. Our recent studies suggest a role for secretory lysosomes in releasing ATP in monocytes. Though poorly defined, targeting ATP release mechanisms in leukocytes have great anti-inflammatory potential.
purinergic signaling; ATP release; inflammation; leukocytes; nucleotide
The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.
GLUT4; IRAP; Rab10; Rab14; adipocytes; TIRF
Mitochondria receive calcium (Ca2+) signals from endoplasmic reticulum (ER) and decode them into pro-apoptotic inputs, which lead to cell death. Therefore, mitochondrial Ca2+ overload is considered a fundamental trigger of the apoptotic process, and several oncogenes and tumor suppressors modify the activity of protein involved in Ca2+ homeostasis to control apoptosis. The identification of the channel responsible for mitochondrial Ca2+ entry, the Mitochondrial Ca2+Uniporter (MCU), together with its regulatory components, MICU1 and MCUR1, provides new molecular tools to investigate this process. Recent data have also shown that miR-25 decreases mitochondrial Ca2+ uptake through selective MCU downregulation, conferring resistance to apoptotic challenges. MCU appears to be downregulated in human colon cancer samples, and accordingly, miR-25 is aberrantly expressed, indicating the importance of mitochondrial Ca2+ regulation in cancer cell survival.
mitochondria; calcium (Ca2+); Mitochondrial Calcium Uniporter (MCU); MicroRNAs (MiRNA); apoptosis; cell death; cancer
Behavioral differences observed between wild communities of the same species have been called “cultures” by some researchers who aimed to underline the similarities with human cultures. However, whether these differences truly result from social learning processes is debated. Despite promising recent research, data acquired in the wild still fail to exclude genetic and ecological factors from being potential explanations for the observed behavioral differences. A potential way to address this problem is through field experiments where communities of the same subspecies are exposed to identical apparatuses. This way, genetic and ecological factors can be controlled for, although their influence cannot be fully excluded. Working with wild-born Sumatran orangutans originating from two genetically distinct populations, we recently combined field experiments with captive work to show that genetic differences could not account for differences in their knowledge of stick use. Additionally, we found evidence that our subjects arrived at the sanctuary with a knowledge that they acquired but could not express in their community of origin. These findings suggest that animal cultures must also be analyzed at the cognitive level. Only in this way can we understand the true extent of animal cultures and how they relate to human cultures.
animal culture; tool use; orangutan; Pongo abelii; mental representations; evolution of culture
The cells that are responsible for detecting magnetic fields in animals remain undiscovered. Previous studies have proposed that pigeons employ a magnetic sense system that consists of six bilateral patches of magnetite containing dendrites located in the rostral subepidermis of the upper beak. We have challenged this hypothesis arguing that clusters of iron-rich cells in this region are macrophages, not magnetosensitive neurons. Here we present additional data in support of this conclusion. We have undertaken high resolution anatomical mapping of iron-rich cells in the rostral upper beak of pigeons, excluding the possibility that a conserved six-loci magnetic sense system exists. In addition we have extended our immunohistochemical studies to a second cohort of pigeons, confirming that iron rich cells in the upper beak are positive for MHCII and CD44, which are expressed by macrophages. We argue that it is important to critically assess conclusions that have been made in the past, while keeping an open mind as the search for the magnetoreceptor continues.
magnetite; magnetoreception; pigeons; anatomical mapping; navigation
The Ras GTPases comprising three main isoforms H-, N- and K-Ras operate at the plasma membrane as molecular switches in essential signaling pathways. Active concentration of the minor phospholipid phosphatidylserine in the inner leaflet of the plasma membrane contributes to the electrostatic potential that is required for K-Ras anchoring to the plasma membrane. We recently observed that staurosporine and related analogs: 7-oxostaurosporine, UCN-01 and UCN-02, long known as relatively non-specific protein kinase inhibitors, block endosomal sorting and recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine to endosomes and endomembranes with concomitant mislocalization of K-Ras. Staurosporines are therefore a new tool to study phosphatidylserine trafficking. We discuss whether the mechanism of action of UCN-01, an FDA-approved staurosporine analog used as an anti-cancer therapeutic, is related to effects on phosphatidylserine subcellular distribution. Given the high prevalence of expression of constitutively active K-Ras in human cancers, we ask whether inhibitors of phosphatidylserine trafficking may have important therapeutic applications.
Ras GTPase; phosphatidylserine; lipid recycling; staurosporine; UCN-01
The dynamic turnover of the L1 cell adhesion molecule to and from the plasma membrane that is mediated through exo-and endocytic trafficking is central to axon outgrowth. Although the ubiquitination of L1 in response to incubation with an L1 antibody that mimics L1-L1 homophilic binding has been previously shown, the endocytic trafficking pathway of the ubiquitinated L1 destined for degradation is yet unclear. I have recently shown that the ubiquitinated L1 is endocytosed by Rabex-5, which is an ubiquitin-binding protein and guanine nucleotide exchange factor for Rab5, into early endosomes from the plasma membrane. Here, I speculate on the putative ubiquitination site within the membrane-proximal ezrin-binding motif in the cytoplasmic domain of L1 and discuss the regulatory role of this motif in the competition between ubiquitination and the binding of ezrin prior to L1 internalization.
endocytosis; cell adhesion molecule; L1; ubiquitin; ezrin
We previously reported that band-pass filtered noise (BFN, bandwidth 20 kHz) affected the echolocation behavior of horseshoe bats in different ways depending on which frequencies within the bats’ hearing range BFN was centered. We found that call amplitudes only increased when BFN was centered on the dominant frequency of the bats' calls. In contrast, call frequencies were shifted for all BFN stimuli centered on or below the dominant frequency of echolocation calls including when BFN was centered at 20 kHz (BFN20), which is far below the range of the bats’ echolocation call frequencies. In the present study we focused on masking effects of BFN20 and used it as a model stimulus for anthropogenic noise to investigate how this noise affected call parameters of echolocation calls, such as their frequency, amplitude, duration and rate. We show that only call frequencies shifted in response to masking BFN20, whereas all other call parameters tested exhibited no noise-related changes. Our findings provide an empirical basis to quantitatively predict the impact of anthropogenic noise on echolocation behavior in bats. They also emphasize the need for a better understanding of the impact of anthropogenic noise on bioacoustic communication and orientation systems in general.
audio-vocal integration; echolocation; horseshoe bat; Lombard effect; mammal; urban noise; vocalization; acoustic communication
In non-polarized cells, calcium-induced exocytosis of “conventional” lysosomes is important in diverse processes like membrane repair after exposure to pore-forming toxins and clearance of cellular debris. Resealing of torn membranes is especially critical for barrier epithelia that directly interact with pathogens and toxins, which can result in membrane microdisruptions and lesions. However, whether lysosomes participate in membrane repair in polarized epithelia has been an open question. We recently reported that in polarized Madin-Darby canine kidney (MDCK) cells, localized influx of calcium induces lysosomes to fuse with the basolateral membrane. This spatial segregation of exocytosis depends on an intact actin cytoskeleton, membrane cholesterol and restricted distribution of fusion machinery such as the t-SNARE syntaxin 4. Our data show that the polarity of syntaxin 4 (which is regulated by the clathrin adaptor protein AP-1) dictates whether lysosomes parachute down to the basolateral membrane or take a ladder up to the apical membrane. Here, we speculate about additional machinery (such as the lysosomal calcium sensor synaptotagmin VII and the v-SNARE VAMP7) that could be involved in polarized fusion of lysosomes with the epithelial membrane. We also discuss the potential importance of lysosome exocytosis in maintaining membrane integrity in the retinal pigment epithelium, the primary tissue affected in blinding diseases such as age-related macular degeneration.
polarized epithelia; membrane repair; fusion machinery; SNAREs; synaptotagmins; retinal pigment epithelium; age-related disease
Olfactometer experiments, in which arthropods are given the choice between two or more odor sources to test behavioral preferences, are commonly used in chemical ecology research. Results of such often lead to conclusions on behavior in an ecologically relevant setting. However, it is widely unknown how well these experiments reflect actual behavior in nature. Recently, we used natural insect herbivores of wild lima bean plants to evaluate their behavior in Y-tube olfactometer experiments compared with feeding experiments. We demonstrated that depending on volatile concentration, insect sex significantly determined preference, and that independent of sex, the actual feeding choice of insects depended on defensive short-distance cues, which did not correlate with volatile cues emitted by the plants. Thus, our study shows that olfactory decisions do not reflect actual feeding choice and that olfactometer experiments may only provide a limited and simplified picture of actual decision making by insects.
plant volatiles; feeding experiments; behavior; decision; herbivore; chemical ecology
Synchronized network activity among groups of interconnected cells is essential for diverse functions in the brain. However, most studies have been made on cellular networks in the mature brain when chemical synapses have been formed. Much less is known about the situation earlier in development. When studying neural progenitors derived from embryonic stem cells and neural progenitors from mice embryos, we found networks of gap junction coupled cells with vivid spontaneous non-random calcium (Ca2+) activity driven by electrical depolarization that stimulated cell growth. Network activity was revealed by single-cell live Ca2+ imaging and further analyzed for correlations and network topology. The analysis revealed the networks to have small-world characteristics with scale-free properties. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.
neural progenitors; networks; calcium signaling; stem cells; gap junctions
sexual selection; beauty selection; evolution; natural selection; David Rothenberg; survival of the beautiful; aesthetics; science of beauty
Ribosomes are the nanomachines that synthesize all cellular proteins from mRNA templates. In eukaryotes, ribosomes, which are composed of ribosomal proteins and rRNA, are mainly assembled in the nucleus. Thus, ribosomal proteins require a nuclear transport step from their place of synthesis in the cytoplasm to their site of assembly in the nucleus. Recognition of import substrates is mediated by different types of nuclear localization signals, which are either directly recognized by import receptors or recruited to these via adaptor proteins. The novel transport adaptor Syo1 (Symportin), which is dedicated to the synchronous import of two functionally related ribosomal proteins, has recently been described. In this review, we highlight and discuss these findings in the context of our current knowledge of ribosome assembly and nucleocytoplasmic transport. We propose that nuclear co-import of functionally and topologically linked cargo could be a widespread strategy to streamline assembly of macromolecular complexes in the nucleus.
nuclear import; transport adaptor; nuclear localization signal; ribosome assembly; ribosomal protein; chaperone
In Pseudomonas aeruginosa eDNA is a crucial component essential for biofilm formation and stability. In this study we report that release of eDNA is influenced by the production of phenazine in P. aeruginosa. A ∆phzA-G mutant of P. aeruginosa PA14 deficient in phenazine production generated significantly less eDNA in comparison with the phenazine producing strains. The relationship between eDNA release and phenazine production is bridged via hydrogen peroxide (H2O2) generation and subsequent H2O2 mediated cell lysis and ultimately release of chromosomal DNA into the extracellular environment as eDNA.
Pseudomonas aeruginosa; phenazines; hydrogen peroxide; extracellular DNA
Platelets, as well as regulating blood hemostasis, are an important component of the body’s defense against invading microbial pathogens. We previously reported that platelets protect during malaria infection by binding Plasmodium-infected erythrocytes (IE) and killing the parasite within. More recent studies have now revealed the platelet plasmocidal factor, platelet factor 4 (PF4) and the red cell-expressed Duffy-antigen molecule as the central players in the parasite killing activity of platelets.
Platelet; malaria; Plasmodium falciparum; platelet factor 4; Duffy-antigen; innate immunity
GABA (gamma-aminobutyric acid) mediates cell-to-cell communication in eukaryotes and interspecies communication in host-microbe interactions. Agrobacterium tumefaciens induces the development of plant tumor in which GABA accumulates. Two periplasmic binding proteins Atu2422 and Atu4243 and their appropriate ABC-transporter are involved in the binding and importation of GABA. The structure of the selective GABA-binding Atu4243 reveals a GABA conformation similar to a proposed model of GABA bound to the mammalian GABAC receptor. The A. tumefaciens atu4243 mutant is affected for GABA uptake, aggressiveness on plant host and GABA-induced degradation of the quorum-sensing signal, hence for horizontal transfer of the tumor-inducing plasmid. Here, we report that a de-repression of atu4243 and its co-regulated neighbor genes affect the fitness of A. tumefaciens during tumor colonization. Atu4243-orthologs are present in several species of the Agrobacterium genus. This addendum highlights the recent data on the GABA transport in the A. tumefaciens plant-pathogen.
GABA; TACA; periplasmic binding protein; ABC transporter; GABAc; Agrobacterium; plant tumour; fitness; quorum-sensing
Humans can behave fairly, but can other species? Recently we tested chimpanzees on a classic human test for fairness, the Ultimatum Game, and found that they behaved similarly to humans. In humans, Ultimatum Game behavior is cited as evidence for a human sense of fairness. By that same logic, we concluded that chimpanzees behaved fairly in our recent study. However, we make a distinction between behavior and motivation. Both humans and chimpanzees behaved fairly, but determining why they did so is more challenging.
behavioral economics; chimpanzee; fairness; primate; ultimatum game
Eg5 (kinesin-5) is a highly conserved microtubule motor protein, essential for centrosome separation and bipolar spindle assembly in human cells. Using an “in vitro” evolution approach, we generated human cancer cells that can grow in the complete absence of Eg5 activity. Characterization of these Eg5-independent cells (EICs) led to the identification of a novel pathway for prophase centrosome separation, which depends on nuclear envelope (NE)-associated dynein. Here, we discuss our recent findings and elaborate on the mechanism by which dynein drives centrosome separation.
centrosome separation; dynein; Eg5; resistance; spindle
Stochastic analysis of superresolution microscopy data obtained from receptor trafficking on neurons reveals novel organized molecular assembly generating long range forces. Would that have been possible with single particle tracking? How have we characterized these molecular assemblies?
analysis; nano-domains; potential wells; stochastic; super-resolution