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1.  From all to (nearly) none 
Cellular Logistics  2014;4:e28114.
The five adaptor protein (AP) complexes function in cargo-selection and coat-recruitment stages of vesicular transport in eukaryotic cells. Much of what we know about AP complex function has come from experimental work using Saccharomyces cerevisiae as a model. Here, using a combination of comparative genomic and phylogenetic approaches we provide evolutionary context for the knowledge gained from this model system by searching the genomes of diverse fungi as well as a member of the sister group to all fungi, Fonticula alba, for presence of AP subunits. First, we demonstrate that F. alba contains all five AP complexes; whereas, similar to S. cerevisiae, most fungi retain only AP-1 to 3. As exceptions, the glomeromycete Rhizophagus irregularis maintains a complete AP-4 and chytrid fungi Spizellomyces punctatus and Batrachochytrium dendrobatidis retain partial AP-4 complexes. The presence of AP-4 subunits in diverse fungi suggests that AP-4 has been independently lost up to seven times in the fungal lineage. In addition to the trend of loss in fungi, we demonstrate that the duplication that gave rise to the β subunits of the AP-1 and AP-2 complexes in S. cerevisiae occurred before the divergence of F. alba and Fungi. Finally, our investigation into the AP complement of basal fungi (Microsporidia and Cryptomycota) demonstrates that while the cryptomycete Rozella allomyces contains an adaptin complement similar to other fungi, the extremely reduced Microsporidia retain, at most, a single cryptic AP complex in the absence of clathrin or any other putative AP-associated coat protein.
PMCID: PMC4022609  PMID: 24843829
AP complex; Adaptins; Microsporidia; clathrin mediated endocytosis; evolutionary cell biology; fungal evolution; membrane trafficking
2.  Multipronged interaction of the COG complex with intracellular membranes 
Cellular Logistics  2014;4:e27888.
The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.
PMCID: PMC3948154  PMID: 24649395
COG complex; Golgi; vesicular trafficking; vesicular tethers; intra-Golgi transport; SNARE; p115; COPI; membrane binding
3.  The proteolytic landscape of the yeast vacuole 
Cellular Logistics  2014;4:e28023.
The vacuole in the yeast Saccharomyces cerevisiae plays a number of essential roles, and to provide some of these required functions the vacuole harbors at least seven distinct proteases. These proteases exhibit a range of activities and different classifications, and they follow unique paths to arrive at their ultimate, common destination in the cell. This review will first summarize the major functions of the yeast vacuole and delineate how proteins are targeted to this organelle. We will then describe the specific trafficking itineraries and activities of the characterized vacuolar proteases, and outline select features of a new member of this protease ensemble. Finally, we will entertain the question of why so many proteases evolved and reside in the vacuole, and what future research challenges exist in the field.
PMCID: PMC4022603  PMID: 24843828
protease; S. cerevisiae; hydrolysis; autophagy; endocytosis; metalloprotease; Vps10; CPY; secretory pathway; Pff1
4.  Novel effects of Brefeldin A (BFA) in signaling through the insulin receptor (IR) pathway and regulating FoxO1-mediated transcription 
Cellular Logistics  2014;4:e27732.
Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.
PMCID: PMC4022606  PMID: 24843827
insulin pathway; Brefeldin A (BFA); FoxO1; SIRT-1; GLUT4; intracellular trafficking
5.  A historical perspective on the lateral diffusion model of GTPase activation and related coupling of membrane signaling proteins 
Cellular Logistics  2014;4:e29389.
Aspects of our discovery of lateral diffusion of the G protein coupled receptor (GPCR) rhodopsin and that a single activated rhodopsin can non-covalently catalyze GTP binding to thousands of GTPases per second on rod disk membranes via this diffusion are summarized herein. Rapid GTPase coupling to membrane-bound phosphodiesterase (PDE) further amplifies the signal via cGMP hydrolysis, essential to visual transduction. Important generalizations from this work are that biomembranes can uniquely concentrate, orient for reaction and provide a solvent appropriate to rapid, powerful and appropriately controlled sequential interaction of signaling proteins. Of equal importance to function is timely control and termination of such powerful amplification via receptor phosphorylation (quenching) and arrestin binding. Downstream kinetic modulation by GTPase activating proteins (GAPs) and regulators of G protein signaling (RGS) and related mechanisms as well as limitations set by membrane domain fencing, structural protein binding etc. can be essential in relevant systems.
PMCID: PMC4160331  PMID: 25279248
GTPases/G proteins; GEFs (guanine nucleotide exchange factors); GPCRs (G protein coupled receptors); GAPs (GTPase activating proteins); effectors; ARF; RAS; RAB
6.  Current understanding of signal amplification in phototransduction 
Cellular Logistics  2014;4:e29390.
The studies of visual signal transduction, or phototransduction, have played a pivotal role in elucidating the most general principles of G protein signaling, particularly in regards to the concept of signal amplification, i.e., the process by which activation of a relatively small number of G protein coupled receptors is transformed into a robust downstream signaling event. In this essay, we summarize our current quantitative understanding of this process in living rods of lower and higher vertebrate animals. An integration of biochemical experiments in vitro with electrophysiological recordings from intact rod photoreceptors indicates that the total number of G protein molecules activated in the course of a light response to a single photon is ~16 in the mouse and ~60 in the frog. This further translates into hydrolysis of ~2000 and ~72 000 molecules of cGMP downstream of G protein, respectively, which represents the total degree of biochemical amplification in the phototransduction cascade.
PMCID: PMC4160332  PMID: 25279249
GTPases/G proteins; GEFs (guanine nucleotide exchange factors); GPCRs (G protein coupled receptors); GAPs (GTPase activating proteins); effectors; ARF; RAS; RAB
7.  G Protein-coupled receptors 
Cellular Logistics  2014;4:e29391.
G protein-coupled receptors and heterotrimeric G proteins can diffuse laterally in the plasma membrane such that one receptor can catalyze the activation (GDP/GTP exchange) of multiple G proteins. In some cases, these processes are fast enough to support molecular signal amplification, where a single receptor maintains the activation of multiple G proteins at steady-state. Amplification in cells is probably highly regulated. It depends upon the identities of the G receptor and G protein - some do and some don’t - and upon the activities of GTPase-activating proteins, membrane scaffolds, and other regulatory partners.
PMCID: PMC4160333  PMID: 25279250
GAPs (GTPase activating proteins); GEFs (guanine nucleotide exchange factors); GPCRs (G protein coupled receptors); GTPases/G proteins; effectors
8.  G protein coupled receptor signaling complexes in live cells 
Cellular Logistics  2014;4:e29392.
Classical models of receptor (GPCR) and G protein (Gαβγ) signaling based on biochemical studies have proposed that receptor stimulation results in G protein activation (Gα-GTP) and dissociation of the heterotrimer (Gα-GTP + Gβγ) to regulate downstream signaling events. Unclear is whether or not there exists freely diffusible, activated Gα-GTP on cellular membranes capable of catalytic signal amplification. Recent studies in live cells indicate that GPCRs serve as platforms for the assembly of macromolecular signaling complexes that include G proteins to support a highly efficient and spatially restricted signaling event, with no requirement for full Gα-GTP and Gβγ dissociation and lateral diffusion within the plasma membrane.
PMCID: PMC4160338  PMID: 25279251
GTPases/G proteins; GEFs (guanine nucleotide exchange factors); GPCRs (G protein coupled receptors); GAPs (GTPase activating proteins); effectors; ARF; RAS; RAB
9.  Tracking of the dynamic localization of the Rab-specific HOPS subunits reveal their distinct interaction with Ypt7 and vacuoles 
Cellular Logistics  2014;4:e29191.
Endosomal and vacuole fusion depends on the two homologous tethering complexes CORVET and HOPS. HOPS binds the activated Rab GTPase Ypt7 via two distinct subunits, Vps39 and Vps41. To understand the participation and possible polarity of Vps41 and Vps39 during tethering, we used an in vivo approach. For this, we established the ligand-induced relocalization to the plasma membrane, using the Mon1-Ccz1 GEF complex that activates Ypt7 on endosomes. We then employed slight overexpression to compare the mobility of the HOPS-specific Vps41 and Vps39 subunits during this process. Our data indicate an asymmetry in the Rab-specific interaction of the two HOPS subunits: Vps39 is more tightly bound to the vacuole, and relocalizes the entire vacuole to the plasma membrane, whereas Vps41 behaved like the more mobile subunit. This is due to their specific Rab binding, as the mobility of both subunits was similar in ypt7∆ cells. In contrast, both HOPS subunits were far less mobile if tagged endogenously, suggesting that the entire HOPS complex is tightly bound to the vacuole in vivo. Similar results were obtained for the endosomal association of CORVET, when we followed its Rab-specific subunit Vps8. Our data provide in vivo evidence for distinct Rab specificity within HOPS, which may explain its function during tethering, and indicate that these tethering complexes are less mobile within the cell than previously anticipated.
PMCID: PMC4156483  PMID: 25210650
HOPS; tethering complex; Vps39; Vps41; endosome; vacuole
10.  Kinesin-2 mediates apical endosome transport during epithelial lumen formation 
Cellular Logistics  2014;4:e28928.
Apical lumen formation is a key step during epithelial morphogenesis of tubular organs. Appropriate transport and targeting of apical proteins to the apical membrane initiation site (AMIS) plays a crucial role in establishing a solitary, central lumen. FIP5, a Rab11-interacting protein, is an important regulator that directs apical endosome trafficking along microtubules toward the AMIS during cytokinesis. However, it is unknown which molecular motor(s) transports FIP5-positive apical endosomes during lumen initiation, and how this process is regulated. In this study, we demonstrate that the interaction of FIP5 with the microtubule motor, Kinesin-2, is required for the movement of FIP5-endosomes and delivery of these endosomes from centrosomes to the cleavage furrow during apical lumen initiation. Loss of Kinesin-2 disrupts targeting of apical proteins to the AMIS and results in multiple lumen formation in MDCK cysts. Our data provide more details to the molecular mechanism of FIP5-dependent apical trafficking during apical lumen formation.
PMCID: PMC4024058  PMID: 24843830
AMIS; FIP5; Kinesin-2; apical trafficking; lumen formation; tubulogenesis
11.  An investigation of the effect of membrane curvature on transmembrane-domain dependent protein sorting in lipid bilayers 
Cellular Logistics  2014;4:e29087.
Sorting of membrane proteins within the secretory pathway of eukaryotic cells is a complex process involving discrete sorting signals as well as physico-chemical properties of the transmembrane domain (TMD). Previous work demonstrated that tail-anchored (TA) protein sorting at the interface between the Endoplasmic Reticulum (ER) and the Golgi complex is exquisitely dependent on the length and hydrophobicity of the transmembrane domain, and suggested that an imbalance between TMD length and bilayer thickness (hydrophobic mismatch) could drive long TMD-containing proteins into curved membrane domains, including ER exit sites, with consequent export of the mismatched protein out of the ER. Here, we tested a possible role of curvature in TMD-dependent sorting in a model system consisting of Giant Unilamellar Vesicles (GUVs) from which narrow membrane tubes were pulled by micromanipulation. Fluorescent TA proteins differing in TMD length were incorporated into GUVs of uniform lipid composition or made of total ER lipids, and TMD-dependent sorting and diffusion, as well as the bending rigidity of bilayers made of microsomal lipids, were investigated. Long and short TMD-containing constructs were inserted with similar orientation, diffused equally rapidly in GUVs and in tubes pulled from GUVs, and no difference in their final distribution between planar and curved regions was detected. These results indicate that curvature alone is not sufficient to drive TMD-dependent sorting at the ER-Golgi interface, and set the basis for the investigation of the additional factors that must be required.
PMCID: PMC4156485  PMID: 25210649
endoplasmic reticulum; giant unilamellar vesicles; hydrophobic mismatch; nanotubes; tail-anchored proteins; optical tweezers; bending rigidity
12.  Distinct patterns of phosphatidylserine localization within the Rab11a-containing recycling system 
Cellular Logistics  2014;4:e28680.
The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.
PMCID: PMC4156484  PMID: 25210648
Rab11-FIP; Rab11a; Rab5; Rab7; Rab8a; live cell microscopy; phophatidylserine; structured illumination
13.  Unique and conserved features of the plant ER-shaping GTPase RHD3 
Cellular Logistics  2014;4:e28217.
The architectural integrity of the endoplasmic reticulum (ER) network depends on the function of membrane-associated dynamin-like GTPases that include metazoan atlastins, plant RHD3 and yeast Sey1p. The evidence that these proteins are sufficient to drive membrane fusion of reconstituted proteoliposomes, and that loss-of-function mutations lead to conspicuous ER shape defects indicates that atlastins, RHD3 and Sey1p promote ER membrane fusion. However, complementation experiments in reciprocal loss-of-function backgrounds have also suggested that RHD3 and Sey1p may be not functionally equivalent, supporting that ER fusion mechanisms may be not entirely conserved in eukaryotes. In this Letter, we provide a brief overview of the field as well as evidence that may explain the functional differences of the plant and yeast ER-shaping dynamin-like GTPases.
PMCID: PMC4013103  PMID: 24812592
ER; Root Hair Defective 3 (RHD3); dynamin-like GTPases; Arabidopsis
14.  Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes 
Cellular Logistics  2014;3:e27609.
The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5–13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.
PMCID: PMC4187004  PMID: 25332840
G protein coupled receptors; GTP-binding protein; Heterotrimeric G proteins; Ras superfamily; guanine nucleotide exchange factors
15.  Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat 
Cellular Logistics  2014;3:e27687.
Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it’s binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity.
PMCID: PMC4187009  PMID: 25332841
vesicle tether; vesicle coat complex; Rab GTPase; p115; COPI
16.  Nobel Prize for Cellular Logistics! 
Cellular Logistics  2013;3:e27194.
PMCID: PMC4091106  PMID: 25054086
17.  Centrosomal AKAP350 modulates the G1/S transition 
Cellular Logistics  2013;3(1):e26331.
AKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participate in the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1/S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.
PMCID: PMC3891632  PMID: 24475373
AKAP350; AKAP450; CG-NAP; Cdk2; centrosome; G1/S transition
18.  Variant-specific prion interactions 
Cellular Logistics  2013;3(1):e25698.
Prions are protein conformations that “self-seed” the misfolding of their non-prion iso-forms into prion, often amyloid, conformations. The most famous prion is the mammalian PrP protein that in its prion form causes transmissible spongiform encephalopathy. Curiously there can be distinct conformational differences even between prions of the same protein propagated in the same host species. These are called prion strains or variants. For example, different PrP variants are faithfully transmitted during self-seeding and are associated with distinct disease characteristics. Variant-specific PrP prion differences include the length of the incubation period before the disease appears and the deposition of prion aggregates in distinct regions of the brain.1 Other more common neurodegenerative diseases (e.g., Alzheimer disease, Parkinson disease, type 2 diabetes and ALS) are likewise caused by the misfolding of a normal protein into a self-seeding aggregate.2-4 One of the most important unanswered questions is how the first prion-like seed arises de novo, resulting in the pathological cascade.
PMCID: PMC3891757  PMID: 24475372
yeast; prion; [PIN+]; [PSI+]
19.  Mitochondrial metabolism as a regulator of keratinocyte differentiation 
Cellular Logistics  2013;3(1):e25456.
Mitochondrial metabolism has traditionally been thought of as a source of cellular energy in the form of ATP. The recent renaissance in the study of cellular metabolism, particularly in the cancer field, has highlighted the fact that mitochondria are also critical biosynthetic and signaling hubs, making these organelles key governors of cellular outcomes.1-5 Using the epidermis as a model system, our recent study looked into the role that mitochondrial metabolism and ROS production play in cellular differentiation in vivo.6 We showed that conditional deletion of the mitochondrial transcription factor, TFAM within the basal cells of the epidermis results in loss of mitochondrial ROS production and impairs epidermal differentiation and hair growth. We demonstrated that mitochondrial ROS generation is required for the propagation of Notch and β-catenin signals which promote epidermal differentiation and hair follicle development respectively. This study bolsters accumulating evidence that oxidative mitochondrial metabolism plays a causal role in cellular differentiation programs. It also provides insights into the role that mitochondrial oxidative signaling plays in a cell type-dependent manner.
PMCID: PMC3891634  PMID: 24475371
mitochondrial metabolism; ATP; reactive oxygen species; TFAM; cellular differentiation
20.  Common flora and intestine 
Cellular Logistics  2013;3(1):e24975.
Commensal microflora engages in a symbiotic relationship with their host, and plays an important role in the development of colorectal cancer (CRC). Pathogenic bacteria promote chronic intestinal inflammation and accelerate tumorigenesis. In sporadic CRC, loss of an effective epithelial barrier occurs at early stage of CRC development. As a result, non-pathogenic bacteria and/or their products infiltrate tumor stroma, drive “tumor-elicited inflammation” and promote CRC progression by activating tumor-associated myeloid and immune cells that produce IL-23 and IL-17. In this article we will summarize the recent advances in understanding the relationship between gut flora and CRC.
PMCID: PMC3906427  PMID: 24516778
IL-17; IL-23; colorectal cancer; commensal flora; epithelial barrier; inflammation
21.  Translational control and autism-like behaviors 
Cellular Logistics  2013;3(1):e24551.
Autism spectrum disorders (ASD) consist of a spectrum of neurodevelopmental diseases with three salient features: reduced social interactions, impaired communication and repetitive/stereotyped behaviors. In a recent study we found that increased eIF4E (eukaryotic initiation factor 4E)-dependent protein synthesis as a result of genetic deletion of Eif4ebp2 (eIF4E-binding protein 2) in mice, stimulates the production of neuroligins (Nlgns, synaptic cell-adhesion molecules important for synapse regulation) and engenders an imbalance of excitatory to inhibitory synaptic transmission (E/I) in CA1 pyramidal neurons. This imbalance is accompanied with deficits in social interaction, communication and repetitive/stereotyped behaviors in Eif4ebp2−/− mice. Using a compound that blocks cap-dependent translation or by knocking down Nlgn1, we restored the E/I balance and reversed the autism-like social deficits.
PMCID: PMC3906422  PMID: 24516777
translational control; ASD; excitation-inhibition balance; autism-like behaviors; mouse models
22.  A note from the Editor-in-Chief 
Cellular Logistics  2012;2(4):175.
PMCID: PMC3607618  PMID: 23536919
23.  Computational method for calculating fluorescence intensities within three-dimensional structures in cells 
Cellular Logistics  2012;2(4):176-188.
The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.
PMCID: PMC3607619  PMID: 23538475
image analysis; microscopy; immunofluorescence; confocal microscopy; wide field; membrane traffic; quantification; isosurface
24.  A comprehensive strategy to identify stoichiometric membrane protein interactomes 
Cellular Logistics  2012;2(4):189-196.
There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.
PMCID: PMC3607620  PMID: 23676845
membrane protein; Ano1; interactome; SILAC; epithelia; salivary gland
25.  The dark face of AMPK as an essential tumor promoter 
Cellular Logistics  2012;2(4):197-202.
Numerous studies have shown that supraphysiological activation of AMPK could inhibit tumor growth. On the other hand, accumulating data also suggest that AMPK activity is required for tumor growth and migration. These findings suggest that physiological activation of AMPK is critical for tumor growth/migration, possibly through maintenance of ATP levels. Our recent study provides the first evidence that the maintenance of cellular NADPH homeostasis is the predominant mechanism by which AMPK promotes tumor cell survival and solid tumor formation. We showed that AMPK activation is required to maintain intracellular NADPH levels through the activation of fatty acid oxidation (FAO) or the inhibition of fatty acid synthesis (FAS) during glucose deprivation or matrix detachment respectively. Through these processes AMPK activation inhibits the rise in reactive oxygen species (ROS) levels and promotes metabolic adaptation in response to metabolic stress. This finding also provides a new therapeutic opportunity through targeting metabolic adaptation of cancer cells, either alone or in combination with conventional anti-cancer drugs that cause metabolic stress.
PMCID: PMC3607621  PMID: 23676995
AMPK; LKB1; CaMKK2; ACC; NADPH; ROS; fatty acids; metabolic stress; cancer

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