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1.  ROCK1 and ROCK2 inhibition alters dendritic spine morphology in hippocampal neurons 
Cellular Logistics  2016;5(4):e1133266.
Communication among neurons is mediated through synaptic connections between axons and dendrites, and most excitatory synapses occur on actin-rich protrusions along dendrites called dendritic spines. Dendritic spines are structurally dynamic, and synapse strength is closely correlated with spine morphology. Abnormalities in the size, shape, and number of dendritic spines are prevalent in neurologic diseases, including autism spectrum disorders, schizophrenia, and Alzheimer disease. However, therapeutic targets that influence spine morphology are lacking. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are potent regulators of the actin cytoskeleton and highly promising drug targets for central nervous system disorders. In this report, we addressed how pharmacologic inhibition of ROCK1 and ROCK2 affects dendritic spine morphology. Hippocampal neurons were transfected with plasmids expressing fluorescently labeled Lifeact, a small actin binding peptide, and then incubated with or without Y-27632, an established pan-ROCK small molecule inhibitor. Using an automated 3D spine morphometry analysis method, we showed that inhibition of ROCK1 and ROCK2 significantly increased the mean protrusion density and significantly reduced the mean protrusion width. A trending increase in mean protrusion length was observed following Y-27632 treatment, and novel effects were observed among spine classes. Exposure to Y-27632 significantly increased the number of filopodia and thin spines, while the numbers of stubby and mushroom spines were similar to mock-treated samples. These findings support the hypothesis that pharmacologic inhibition of ROCK1 and ROCK2 may convey therapeutic benefit for neurologic disorders that feature dendritic spine loss or aberrant structural plasticity.
PMCID: PMC4820816  PMID: 27054047
Automated image analysis; Dendritic spine morphology; Hippocampal neurons; RhoA; Rho kinase; ROCK1; ROCK2
2.  Arf-like GTPase Arl8: Moving from the periphery to the center of lysosomal biology 
Cellular Logistics  2015;5(3):e1086501.
Lysosomes are dynamic organelles that not only mediate degradation of cellular substrates but also play critical roles in processes such as cholesterol homeostasis, plasma membrane repair, antigen presentation, and cell migration. The small GTPase Arl8, a member of Arf-like (Arl) family of proteins, has recently emerged as a crucial regulator of lysosome positioning and membrane trafficking toward lysosomes. Through interaction with its effector SKIP, the human Arl8 paralog (Arl8b) mediates kinesin-1 dependent motility of lysosomes on microtubule tracks toward the cell periphery. Arl8b-mediated kinesin-driven motility is also implicated in regulating lytic granule polarization in NK cells, lysosome tubulation in macrophages, cell spreading, and migration. Moreover, Arl8b regulates membrane traffic toward lysosomes by recruiting subunits of the HOPS complex, a multi-subunit tethering complex that mediates endo-lysosome fusion. Here we provide a brief review on this recently characterized lysosomal GTPase and summarize the studies focusing on its known functions in regulating lysosomal motility and delivery of endocytic cargo to the lysosomes. We also explore the role of human Arl8b and its orthologs upon infection by intracellular pathogens.
PMCID: PMC4820812  PMID: 27057420
autophagy; Arf-like GTPase; Arl8; BORC; HOPS; late endosome; lysosome; membrane trafficking; Motors; SKIP
3.  Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in Saccharomyces cerevisiae 
Cellular Logistics  2016;6(3):e1228791.
NEO1 is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive neo1ts alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of neo1-2ts growth defects yielded YPT7, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion. YPT7 suppressed the vacuole fragmentation phenotype of neo1-2, but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion in vitro and is a potential transport substrate of Neo1. Strains deficient in PE synthesis (psd1Δ psd2Δ) displayed fragmented vacuoles and the neo1-2 fragmented vacuole phenotype was also suppressed by overexpression of PSD2, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast, neo1-2 was not suppressed by overexpression of VPS39, an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane.
PMCID: PMC5058351  PMID: 27738552
flippase; membrane asymmetry; P4-ATPase; Rab protein; SNARE; vacuoles; Ypt7
4.  Diacylglycerol kinases in membrane trafficking 
Cellular Logistics  2015;5(2):e1078431.
Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years.
PMCID: PMC4820814  PMID: 27057419
diacylglycerol; diacylglycerol kinase; endocytic recycling; Golgi; membrane trafficking; MICAL-L1; phosphatidic acid
5.  Role of the epithelial cell-specific clathrin adaptor complex AP-1B in cell polarity 
Cellular Logistics  2015;5(2):e1074331.
Epithelial cells are important for organ development and function. To this end, they polarize their plasma membrane into biochemically and physically distinct membrane domains. The apical membrane faces the luminal site of an organ and the basolateral domain is in contact with the basement membrane and neighboring cells. To establish and maintain this polarity it is important that newly synthesized and endocytic cargos are correctly sorted according to their final destinations at either membrane. Sorting takes place at one of 2 major sorting stations in the cells, the trans-Golgi network (TGN) and recycling endosomes (REs). Polarized sorting may involve epithelial cell-specific sorting adaptors like the AP-1B clathrin adaptor complex. AP-1B facilitates basolateral sorting from REs. This review will discuss various aspects of basolateral sorting in epithelial cells with a special emphasis on AP-1B.
PMCID: PMC4820817  PMID: 27057418
recylcing endosomes; AP-1B; epithelial cells; cell polarity; basolateral membrane
6.  An improved reversibly dimerizing mutant of the FK506-binding protein FKBP 
Cellular Logistics  2016;6(3):e1204848.
FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.
PMCID: PMC5058350  PMID: 27738551
dimerization; F36M; FKBP; FK506-binding protein; regulatable secretory cargo
7.  Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis 
Cellular Logistics  2015;5(1):e1025182.
Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.
PMCID: PMC4501207  PMID: 26196023
exocytosis; Golgi apparatus; SNARE; sphingolipids; TI-VAMP/VAMP7; BFA, Brefeldin A; Cer, Ceramide; ER, Endoplasmic Reticulum; GlcCer, Glucosylceramide; GM3, ganglioside monosialic acid 3; GPI, Glycosylphosphatidylinositol; GSL, Glycosphingolipids; LC, Long Chain; PI, Phosphatidylinositide; PM, Plasma Membrane; SM, Sphingomyelin; TGN, = Trans-Golgi Network; TI-VAMP/VAMP7, Tetanus neurotoxin-insensitive vesicle-associated membrane protein / Vesicle associated membrane protein 7; VLC, very long vhain; VSVG, Vesicular Stomatitis Virus Glycoprotein
8.  An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster  
Cellular Logistics  2015;5(1):e1023423.
Transcription activator-like effector nucleases (TALENs) emerged as powerful tools for locus-specific genome engineering. Due to the ease of TALEN assembly, the key to streamlining TALEN-induced mutagenesis lies in identifying efficient TALEN pairs and optimizing TALEN mRNA injection concentrations to minimize the effort to screen for mutant offspring. Here we present a simple methodology to quantitatively assess bi-allelic TALEN cutting, as well as approaches that permit accurate measures of somatic and germline mutation rates in Drosophila melanogaster. We report that percent lethality from pilot injection of candidate TALEN mRNAs into Lig4 null embryos can be used to effectively gauge bi-allelic TALEN cutting efficiency and occurs in a dose-dependent manner. This timely Lig4-dependent embryonic survival assay also applies to CRISPR/Cas9-mediated targeting. Moreover, the somatic mutation rate of individual G0 flies can be rapidly quantitated using SURVEYOR nuclease and capillary electrophoresis, and germline transmission rate determined by scoring progeny of G0 outcrosses. Together, these optimized methods provide an effective step-wise guide for routine TALEN-mediated gene editing in the fly.
PMCID: PMC4501208  PMID: 26196022
engineered endonuclease; genome engineering; mutagenesis; screening; TALEN; TALENs, Transcription activator-like effector nucleases; TALEs, TAL effectors; ZFNs, Zinc Finger Nucleases; CRISPR, Clustered Regularly Interspersed Short Palindromic Repeats; Cas9, CRISPR-associated; RVDs, repeat-variable diresidues; DSBs, double-stranded breaks; NHEJ, non-homologous end joining; HR, homologous recombination; RFLP, restriction fragment length polymorphism; HRMA, high resolution melt analysis.
9.  Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins 
Cellular Logistics  2014;4(4):e986399.
The mitochondria-associated membrane (MAM) is an endoplasmic reticulum (ER) domain that forms contacts with mitochondria and accommodates Ca2+ transfer between the two organelles. The GTPase Rab32 regulates this function of the MAM via determining the localization of the Ca2+ regulatory transmembrane protein calnexin to the MAM. Another function of the MAM is the regulation of mitochondrial dynamics mediated by GTPases such as dynamin-related protein 1 (Drp1). Consistent with the importance of the MAM for mitochondrial dynamics and the role of Rab32 in MAM enrichment, the inactivation of Rab32 leads to mitochondrial collapse around the nucleus. However, Rab32 and related Rabs also perform intracellular functions at locations other than the MAM including melanosomal trafficking, autophagosome formation and maturation, and retrograde trafficking to the trans-Golgi network (TGN). This plethora of functions raises questions concerning the original cellular role of Rab32 in the last common ancestor of animals and its possible role in the last eukaryotic common ancestor (LECA). Our results now shed light on this conundrum and identify a role in Drp1-mediated mitochondrial dynamics as one common denominator of this group of Rabs, which includes the paralogues Rab32A and Rab32B, as well as the more recently derived Rab29 and Rab38 proteins. Moreover, we provide evidence that this mitochondrial function is dictated by the extent of ER-association of Rab32 family proteins.
PMCID: PMC4355727  PMID: 25767741
Drp1; Mitochondrial dynamics; Rab32 Rab38; Rab29
11.  Ypt/Rab GTPases regulate two intersections of the secretory and the endosomal/lysosomal pathways 
Cellular Logistics  2014;4(3):e954870.
A prevailing question in the Ypt/Rab field is whether these conserved GTPases are specific to cellular compartments. The established role for Ypt1 and its human homolog Rab1 is in endoplasmic reticulum (ER)-to-Golgi transport. More recently these regulators were implicated also in autophagy. Two different TRAPP complexes, I and III, were identified as the guanine-nucleotide-exchange factors (GEFs) of Ypt1 in ER-to-Golgi transport and autophagy, respectively. Confusingly, Ypt1 and TRAPP III were also suggested to regulate endosome-to-Golgi transport, implying that they function at multiple cellular compartments, and bringing into question the nature of Ypt/Rab specificity. Recently, we showed that the role of TRAPP III and Ypt1 in autophagy occurs at the ER and that they do not regulate endosome-to-Golgi transport. Here, we discuss the significance of this conclusion to the idea that Ypt/Rabs are specific to cellular compartments. We postulate that Ypt1 regulates 2 alternative routes emanating from the ER toward the Golgi and the lysosome/vacuole. We further propose that the secretory and endocytic/lysosomal pathways intersect in 2 junctures, and 2 Ypts, Ypt1 and Ypt31, coordinate transport in the 2 intersections: Ypt1 links ER-to-Golgi and ER-to-autophagy transport, whereas Ypt31 links Golgi-to-plasma membrane (PM) transport with PM-to-Golgi recycling through endosomes.
PMCID: PMC4279804  PMID: 25610722
cellular recycling; ER-phagy; endosome-to-Golgi transport; GTPase; Rab; secretory pathway; TRAPP; Ypt1; Ypt31
12.  AKAP350C targets to mitochondria via a novel amphipathic alpha helical domain 
Cellular Logistics  2014;4(3):e943597.
Mitochondria regulate metabolism and homeostasis within cells. Mitochondria are also very dynamic organelles, constantly undergoing fission and fusion. The importance of maintaining proper mitochondrial dynamics is evident in the various diseases associated with defects in these processes. Protein kinase A (PKA) is a key regulator of mitochondrial dynamics. PKA is spatially regulated by A-Kinase Anchoring Proteins (AKAPs). We completed cloning of a novel AKAP350 isoform, AKAP350C. Immunostaining for endogenous AKAP350C showed localization to mitochondria. The carboxyl-terminal 54-amino acid sequence unique to AKAP350C contains a novel amphipathic alpha helical mitochondrial-targeting domain. AKAP350C co-localizes with Mff (mitochondrial fission protein) and mitofusins 1 and 2 (mitochondrial fusion proteins), and likely regulates mitochondrial dynamics by scaffolding PKA and mitochondrial fission and fusion proteins.
PMCID: PMC4279805  PMID: 25610720
AKAP; AKAP350; AKAP9; CG-NAP; AKAP450; mitochondria; mitofusin; Mff; AKAP350, A-kinase anchoring protein 350; Mff, Mitochondrial fission protein; Mfn1, Mitofusin 1; Mfn2, Mitofusin 2
13.  Class C ABC transporters and Saccharomyces cerevisiae vacuole fusion 
Cellular Logistics  2014;4(3):e943588.
Membrane fusion is carried out by core machinery that is conserved throughout eukaryotes. This is comprised of Rab GTPases and their effectors, and SNARE proteins, which together are sufficient to drive the fusion of reconstituted proteoliposomes. However, an outer layer of factors that are specific to individual trafficking pathways in vivo regulates the spatial and temporal occurrence of fusion. The homotypic fusion of Saccharomyces cerevisiae vacuolar lysosomes utilizes a growing set of factors to regulate the fusion machinery that include members of the ATP binding cassette (ABC) transporter family. Yeast vacuoles have five class C ABC transporters that are known to transport a variety of toxins into the vacuole lumen as part of detoxifying the cell. We have found that ABCC transporters can also regulate vacuole fusion through novel mechanisms. For instance Ybt1 serves as negative regulator of fusion through its effects on vacuolar Ca2+ homeostasis. Additional studies showed that Ycf1 acts as a positive regulator by affecting the efficient recruitment of the SNARE Vam7. Finally, we discuss the potential interface between the translocation of lipids across the membrane bilayer, also known as lipid flipping, and the efficiency of fusion.
PMCID: PMC4292212  PMID: 25610719
SNARE; PI3P; Vam7; Ycf1; Ybt1; Nft1; Vmr1; Bpt1; Ca2+ homeostasis; lipid flipping; ABC, ATP binding cassette; DAG, diacylglycerol; HOPS, homotypic fusion and vacuole protein sorting complex; MDR, multidrug resistance; MSD, membrane spanning domain; NBD, nucleotide binding domain; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3, 5)P2, phosphatidylinositol 3, 5-bisphosphate; PS, phosphatidylserine; PX, phox homology; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors
14.  TGN exit of the cation-independent mannose 6-phosphate receptor does not require acid hydrolase binding 
Cellular Logistics  2014;4(3):e954441.
The cation-independent mannose 6-phosphate (Man-6-P) receptor (CI-MPR) binds newly synthesized, Man-6-P-containing lysosomal acid hydrolases in the trans-Golgi network (TGN) for clathrin-mediated transport to endosomes. It has remained unresolved, however, whether acid hydrolase binding is required for exit of the CI-MPR from the TGN. To address this question we used a B cell line derived from a Mucolipidosis type II (MLII)/I-cell disease patient. In MLII patients, acid hydrolases do not acquire the Man-6-P recognition marker and as a consequence do not bind to the CI-MPR. This causes secretion of the majority of the acid hydrolases and a decreased lysosomal activity resulting in typical inclusion bodies. In agreement herewith, ultrastructural analysis of the MLII patient derived B cells showed numerous inclusion bodies with undigested material, which we defined as autolysosomes. By quantitative immuno-electron microscopy we then studied the distribution of the CI-MPR in these cells. We found that the level of co-localization of TGN-localized CI-MPR and clathrin was similar in MLII and control B cells. Moreover, the CI-MPR was readily found in endosomes of MLII cells and the TGN-to-early endosome ratio of CI-MPR labeling was unaltered. These data show that there is no block in TGN exit of the CI-MPR in the absence of Man-6-P-modified acid hydrolases. Notably, late endosomes and inclusion bodies in MLII B cells contained increased levels of the CI-MPR, which likely reflects the reduced degradative capacity of these compartments.
PMCID: PMC4292573  PMID: 25610721
B cells; cation-independent mannose 6-phosphate receptor; electron microscopy; lysosomal acid hydrolases; mannose 6-phosphate modification; Mucolipidosis type II; trans-Golgi network; CI-MPR, cation-independent MPR; FBS, fetal bovine serum; GA, glutaraldehyde; IGF-II, insulin-like growth factor II; MLII, Mucolipidosis type II; MPR, mannose 6-phosphate receptor; PB, phosphate buffer; PFA, paraformaldehyde; TGN, trans-Golgi network
15.  Is the model of signal amplification by GPCRs/GEFs activating multiple GTPases relevant to a broad spectrum of heterotrimeric and RAS superfamily GTPases? 
Cellular Logistics  2014;4(2):e943602.
Concepts or models of biological processes shape how we think about them, discuss them, and design experiments to test aspects of them. Because of the importance of our models of cell signaling by regulatory GTPases and the desire to extend those models to related signaling modules, I have throughout my career been fascinated by the similarities and differences between the modeling of heterotrimeric G protein and monomeric RAS superfamily GTPases. Recent discussions with colleagues led me to conclude that there is a growing divergence in how researchers model the activation and signaling processes of monomeric and trimeric GTPases and also a surprising lack of consensus within each camp. This series of articles arose in response to these discussions and is intended to spark new ones.
PMCID: PMC4276334  PMID: 25610716
ADP-ribosylation factor (ARF); GTPase activating protein (GAP); G-protein coupled receptor (GPCR); Guanine nucleotide exchange factor (GEF); Receptor tyrosine kinase (RTK)
16.  GEF-effector interactions 
Cellular Logistics  2014;4(2):e943616.
Members of the Arf family of small GTP-binding proteins, or GTPases, are activated by guanine nucleotide exchange factors (GEFs) that catalyze GDP release from their substrate Arf, allowing GTP to bind. In the secretory pathway, Arf1 is first activated by GBF1 at the cis-Golgi, then by BIG1 and BIG2 at the trans-Golgi and trans-Golgi network (TGN). Upon activation, Arf1-GTP interacts with effectors such as coat complexes, and is able to recruit different coat complexes to different membrane sites in cells. The COPI coat is primarily recruited to cis-Golgi membranes, whereas other coats, such as AP-1/clathrin, and GGA/clathrin, are recruited to the trans-Golgi and the TGN. Although Arf1-GTP is required for stable association of these various coats to membranes, and is sufficient in vitro, other molecules, such as vesicle cargo and coat receptors on the membrane, contribute to specificity of coat recruitment in cells. Another mechanism to achieve specificity is interaction of effectors such as coats with the GEF itself, which would increase the concentration of a given coat in proximity to the site where Arf is activated, thus favoring its recruitment. This interaction between a GEF and an effector could also provide a mechanism for spatial organization of vesicle budding sites, similar to that described for Cdc42-mediated establishment of polarity sites such as the emerging bud in yeast. Another factor affecting the amount of freely diffusible Arf1-GTP in membranes is the GEF(s) themselves acting as effectors. Sec7p, the yeast homolog of mammalian BIG1 and BIG2, and Arno/cytohesin 2, a PM-localized Arf1 GEF, both bind to Arf1-GTP. This binding to the products of the exchange reaction establishes a positive feedback loop for activation.
PMCID: PMC4279777  PMID: 25610717
ADP-ribosylation factor (Arf); Cdc42; Golgi Brefeldin A resistant guanine nucleotide exchange Factor 1 (GBF1); Guanine nucleotide exchange factor (GEF); Sec7; Turing-type mechanism; vesicle budding
17.  Spatial and temporal control of Rho GTPase functions 
Cellular Logistics  2014;4(2):e943618.
Rho family GTPases control almost every aspect of cell physiology and, since their discovery, a wealth of knowledge has accumulated about their biochemical regulation and function. However, each Rho GTPase distributes between multiple cellular compartments, even within the same cell, where they are controlled by multiple regulators and signal to multiple effectors. Thus, major questions about spatial and temporal aspects of regulation remain unanswered. In particular, what are the nano-scale dynamics for their activation, membrane targeting, diffusion, effector activation and GTPase inactivation? How do these mechanisms differ in the different cellular compartments where Rho GTPases function? Addressing these complex aspects of Rho GTPase biology will significantly advance our understanding of the spatial and temporal control of cellular functions.
PMCID: PMC4279778  PMID: 25610718
Rho GTPases; Rac; GEFs (guanine nucleotide exchange factors); GAPs (GTPase activating proteins); RhoGDI (Rho GDP-dissociation inhibitor); effectors; prenylation; S-palmitoylation; lipid rafts; supported bilayers; domain boundaries; diffusion
18.  Altered trafficking of mutated growth factor receptors and their associated molecules 
Cellular Logistics  2014;4:e28461.
Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.
PMCID: PMC4156482  PMID: 25210647
endocytosis; endosomes; lysosomes; membrane vesicles; oncogenesis; receptor tyrosine kinase; signal transduction; ubiquitination
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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