RESUMO
Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrina alfaVbeta3/metabolismo , Actinas/genética , Animais , Linhagem Celular , Movimento Celular/fisiologia , Citoesqueleto/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Adesões Focais/genética , Integrina alfaVbeta3/genética , CamundongosRESUMO
Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.
Assuntos
Técnicas Citológicas/métodos , Animais , Anticorpos Biespecíficos , Linfócitos B/imunologia , Células COS , Linhagem Celular , Sobrevivência Celular , Chlorocebus aethiops , Técnicas Citológicas/instrumentação , Testes Imunológicos de Citotoxicidade/instrumentação , Testes Imunológicos de Citotoxicidade/métodos , Células HEK293 , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoterapia , Células K562 , Ativação Linfocitária , Neoplasias/imunologia , Neoplasias/terapia , Ligação Proteica , Linfócitos T Citotóxicos/imunologia , Células U937RESUMO
Lactate, an intermediary between glycolysis and mitochondrial oxidative phosphorylation, reflects the metabolic state of neurons. Here, we utilized a genetically-encoded lactate FRET biosensor to uncover subpopulations of distinct metabolic states among Drosophila glutamatergic neurons. Neurons within specific subpopulations exhibited correlated lactate flux patterns that stemmed from inherent cellular properties rather than neuronal interconnectivity. Further, individual neurons exhibited consistent patterns of lactate flux over time such that stimulus-evoked changes in lactate were correlated with pre-treatment fluctuations. Leveraging these temporal autocorrelations, deep-learning models accurately predicted post-stimulus responses from pre-stimulus fluctuations. These findings point to the existence of distinct neuronal subpopulations, each characterized by unique lactate dynamics, and raise the possibility that neurons with correlated metabolic activities might synchronize across different neural circuits. Such synchronization, rooted in neuronal metabolic states, could influence information processing in the brain.
RESUMO
The mitochondrial permeability transition pore (mPTP) is a supramolecular channel that regulates exchange of solutes across cristae membranes, with executive roles in mitochondrial function and cell death. The contribution of the mPTP to normal physiology remains debated, although evidence implicates the mPTP in mitochondrial inner membrane remodeling in differentiating progenitor cells. Here, we demonstrate that strict control over mPTP conductance shapes metabolic machinery as cells transit toward hematopoietic identity. Cells undergoing the endothelial-to-hematopoietic transition (EHT) tightly control chief regulatory elements of the mPTP. During EHT, maturing arterial endothelium restricts mPTP activity just prior to hematopoietic commitment. After transition in cellular identity, mPTP conductance is restored. In utero treatment with NIM811, a molecule that blocks sensitization of the mPTP to opening by Cyclophilin D (CypD), amplifies oxidative phosphorylation (OXPHOS) in hematopoietic precursors and increases hematopoiesis in the embryo. Additionally, differentiating pluripotent stem cells (PSCs) acquire greater organization of mitochondrial cristae and hematopoietic activity following knockdown of the CypD gene, Ppif. Conversely, knockdown of Opa1, a GTPase critical for proper cristae architecture, induces cristae irregularity and impairs hematopoiesis. These data elucidate a mechanism that regulates mitochondrial maturation in hematopoietic precursors and underscore a role for the mPTP in the acquisition of hematopoietic fate.
Assuntos
Células-Tronco Hematopoéticas , Mitocôndrias , Poro de Transição de Permeabilidade Mitocondrial , Animais , Mitocôndrias/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Hematopoese , Peptidil-Prolil Isomerase F/metabolismo , Peptidil-Prolil Isomerase F/genética , Diferenciação Celular , Fosforilação Oxidativa , Feminino , Camundongos Endogâmicos C57BLRESUMO
The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generate diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that conditional male mice with genetic overexpression of Ndufs4 exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping protein STOML2 in linking NDUFS4 with improved cristae morphology. Together, we provide the evidence on the central role of NDUFS4 as a regulator of cristae remodeling and mitochondrial function in kidney podocytes. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Masculino , Animais , Camundongos , Nefropatias Diabéticas/genética , Diabetes Mellitus Experimental/genética , Membranas Mitocondriais , Rim , Mitocôndrias , Complexo I de Transporte de Elétrons/genéticaRESUMO
Maintaining protein homeostasis is essential for cellular health. During times of proteotoxic stress, cells deploy unique defense mechanisms to achieve resolution. Our previous research uncovered a cross-compartmental Mitochondrial to Cytosolic Stress Response (MCSR), a unique stress response activated by the perturbation of mitochondrial proteostasis, which ultimately results in the improvement of proteostasis in the cytosol. Here, we found that this signaling axis also influences the unfolded protein response of the endoplasmic reticulum (UPR ER ), suggesting the presence of a Mitochondria to ER Stress Response (MERSR). During MERSR, the IRE1 branch of UPR ER is inhibited, introducing a previously unknown regulatory component of MCSR. Moreover, proteostasis is enhanced through the upregulation of the PERK-eIF2a signaling pathway, increasing phosphorylation of eIF2a and improving the ER's capacity to manage greater proteostasis load. MERSR activation in both poly-glutamine (poly-Q) and amyloid-beta (Aß) C. elegans disease models also led to improvement in both aggregate burden and overall disease outcome. These findings shed light on the coordination between the mitochondria and the ER in maintaining cellular proteostasis and provides further evidence for the importance of intercompartmental signaling.
RESUMO
The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generated diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model to investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that these conditional mice exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping proteins in linking NDUFS4 with improved cristae morphology. Taken together, we discover the central role of NDUFS4 as a powerful regulator of cristae remodeling, respiratory supercomplexes assembly, and mitochondrial ultrastructure in vitro and in vivo. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.
RESUMO
The nuclear pore complex (NPC) comprises more than 30 nucleoporins (NUPs) and is a hallmark of eukaryotes. NUPs have been suggested to be important in regulating gene transcription and 3D genome organization. However, evidence in support of their direct roles remains limited. Here, by Cut&Run, we find that core NUPs display broad but also cell-type-specific association with active promoters and enhancers in human cells. Auxin-mediated rapid depletion of two NUPs demonstrates that NUP93, but not NUP35, directly and specifically controls gene transcription. NUP93 directly activates genes with high levels of RNA polymerase II loading and transcriptional elongation by facilitating full BRD4 recruitment to their active enhancers. dCas9-based tethering confirms a direct and causal role of NUP93 in gene transcriptional activation. Unexpectedly, in situ Hi-C and H3K27ac or H3K4me1 HiChIP results upon acute NUP93 depletion show negligible changesS2211-1247(22)01437-1 of 3D genome organization ranging from A/B compartments and topologically associating domains (TADs) to enhancer-promoter contacts.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Poro Nuclear , Genoma , Cromatina , Proteínas de Ciclo Celular/genéticaRESUMO
We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.
Assuntos
Membrana Celular/metabolismo , Integrinas/química , Integrinas/metabolismo , Microscopia/métodos , Regulação Alostérica , Adesão Celular , Movimento Celular , Fibronectinas/metabolismo , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Modelos Biológicos , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Leucócitos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sequência de Aminoácidos , Polarização de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Células Jurkat , Leucócitos/citologia , Antígeno-1 Associado à Função Linfocitária/genética , Microscopia de Fluorescência/métodos , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Yeast cells form a single mating projection when exposed to mating pheromone, a classic example of cell polarity. Prolonged treatment with pheromone or specific mutations results in alternative cell polarity behaviours. The authors performed mathematical modelling to investigate these unusual cell morphologies from the perspective of balancing spatial amplification (i.e. positive feedback that localises components) with spatial tracking (i.e. negative feedback that allows sensing of gradient). First, they used generic models of cell polarity to explore different cell polarity behaviours that arose from changes in the model spatial dynamics. By exploring the positive and negative feedback loops in each stage of a two-stage model, they simulated a variety of cell morphologies including single bending projections, single straight projections, periodic multiple projections and simultaneous double projections. In the second half of the study, they used a two-stage mechanistic model of yeast cell polarity focusing on G-protein signalling to integrate the modelling results more closely with the authors' previously published experimental observations. In summary, the combination of modelling and experiments describes how yeast cells exhibit a diversity of cell morphologies arising from two-stage G-protein signalling dynamics modulated by positive and negative feedbacks.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Polaridade Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Simulação por Computador , Retroalimentação Fisiológica/fisiologiaRESUMO
Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180(o) switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gßγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Peptídeos/farmacologia , Feromônios/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Quimiotaxia/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Acasalamento , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microfluídica , Mutação , Peptídeos/metabolismo , Feromônios/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismoRESUMO
Polarized cell morphogenesis requires actin cytoskeleton rearrangement for polarized transport of proteins, organelles and secretory vesicles, which fundamentally underlies cell differentiation and behavior. During yeast mating, Saccharomyces cerevisiae responds to extracellular pheromone gradients by extending polarized projections, which are likely maintained through vesicle transport to (exocytosis) and from (endocytosis) the membrane. We experimentally demonstrate that the projection morphology is pheromone concentration-dependent, and propose the underlying mechanism through mathematical modeling. The inclusion of membrane flux and dynamically evolving cell boundary into our yeast mating signaling model shows good agreement with experimental measurements, and provides a plausible explanation for pheromone-induced cell morphology.
Assuntos
Endocitose/fisiologia , Exocitose/fisiologia , Feromônios/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Modelos TeóricosRESUMO
Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiaea-cells to sense and respond to spatial gradients of the mating pheromone alpha-factor produced in a microfluidics chamber; the focus was on bar1Delta strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient ( approximately 80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average alpha-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the K(d) of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Delta and ste2(300Delta)) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of alpha-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.
Assuntos
Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Atrativos Sexuais/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Extensões da Superfície Celular/metabolismo , Simulação por Computador , Fator de Acasalamento , Microfluídica , Mutação , Peptídeos/metabolismo , Receptores de Fator de Acasalamento/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
OBJECTIVES: The purpose of this study was to determine how photodynamic therapy (PDT) promotes stabilization and reduction of regional atherosclerosis. BACKGROUND: Photodynamic therapy, a combination of photosensitizer and targeted light to promote cell apoptosis, has been shown to reduce atherosclerotic plaque inflammation. METHODS: Forty New Zealand White rabbits were fed with cholesterol. The iliac arteries were balloon denuded and randomized to receive either PhotoPoint PDT treatment (photosensitizer and light) (Miravant Medical Technologies, Santa Barbara, California), photosensitizer (MV0611) alone, or light alone and were then compared at 7 and 28 days. Arteries were examined for evidence of plaque volume, cell number, macrophage and smooth muscle cell (SMC) content, and plaque cell proliferation. RESULTS: Compared with contralateral iliac artery controls at 7 days, plaque progression was reduced by approximately 35% (p < 0.01); plaque progression was further reduced to approximately 53% (p < 0.01) by 28 days, leading to an increase in lumen patency (p < 0.05). At 7 days after PDT, percent plaque area occupied by macrophages decreased by approximately 98% (p < 0.001) and SMCs by approximately 72% (p < 0.05). At 28 days after PDT, removal of macrophages was sustained (approximately 92% decrease, p < 0.001) and plaques were repopulated with non-proliferating SMCs (approximately 220% increase, p < 0.001). There was no evidence of negative or expansive arterial remodeling, thrombosis, or aneurysm formation. CONCLUSIONS: Photodynamic therapy simultaneously reduces plaque inflammation and promotes repopulation of plaques with a SMC-rich stable plaque cell phenotype while reducing disease progression. These early healing responses suggest that PDT is a promising therapy for the treatment of acute coronary syndromes.
Assuntos
Aterosclerose/tratamento farmacológico , Mesoporfirinas/uso terapêutico , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Aterosclerose/sangue , Aterosclerose/patologia , Colesterol/sangue , Artéria Ilíaca/patologia , Macrófagos/patologia , Masculino , Mesoporfirinas/farmacologia , Miócitos de Músculo Liso/patologia , Fotoquimioterapia/efeitos adversos , Fármacos Fotossensibilizantes/farmacologia , Projetos Piloto , Coelhos , Cicatrização/efeitos dos fármacosRESUMO
The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85alpha, p55alpha, p50alpha, and p85beta. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA-deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T/fisiologia , Animais , Proliferação de Células , Técnicas In Vitro , Cooperação Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositol 3-Quinases/deficiência , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/metabolismo , Subunidades Proteicas , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Serina-Treonina Quinases TORRESUMO
Phosphoinositide 3-kinase (PI3K) is a ubiquitously expressed signaling enzyme that plays an integral role in development and activation of B cells. B cell receptor (BCR)-driven proliferation is completely blocked either in cells lacking the p85alpha regulatory isoform of PI3K or in wild-type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85alpha to early signaling events has not been fully investigated. Here we show that B cells lacking p85alpha have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85alpha results in partial reductions in Ca2+ mobilization and IkappaB phosphorylation, whereas ERK phosphorylation is not diminished. Moreover, although Akt phosphorylation is partially reduced, phosphorylation of several proteins downstream of Akt is preserved. These partial impairments suggest that there are other routes to PI3K activation in B cells apart from p85alpha-associated catalytic subunits. Notably, addition of phorbol ester restores BCR-mediated proliferation in p85alpha-deficient cells but not wild-type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85alpha is a failure to activate diacylglycerol-regulated signaling enzymes, most likely protein kinase C.