Cecilia Alvarez

Autophagosome formation depends on the small GTPase Rab1 and functional ER exit sites.

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.

Dissection of Membrane Dynamics of the ARF‐Guanine Nucleotide Exchange Factor GBF1

ADP‐ribosylation factor (ARF)‐facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine nucleotide exchange factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)–Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP‐ribosylation factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP‐tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t1/2 is approximately 17 ± 1 seconds). GBF1 cycles faster than GFP‐tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF–GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER‐Golgi traffic.

Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons.

Previous inquiries into the effects of Brefeldin A (BFA) have largely concentrated on dynamics of ER-Golgi membrane traffic, predominantly after relatively short treatments with the drug. We have now analyzed the effects of long BFA treatment on overall cell morphology, behavior of resident and cycling Golgi proteins, and microtubular and actin cytoskeletons organization. Prolonged (15 h or 40 h) treatment of normal rat kidney (NRK) cells with BFA caused dramatic swelling of the Endoplasmic Reticulum (ER) and shifted its localization to the periphery of the cells. The Golgi complex was disassembled and Golgi proteins redistributed and persisted in partially distinct compartments. Prolonged BFA treatment resulted in marked disruption of the MT and actin cytoskeleton. Peripheral MT were absent and tubulin staining was concentrated in short astral MT emanating from the microtubule organizing center (MTOC). Actin stress fibers were largely absent and actin staining was concentrated within a perinuclear area. Within this region, actin localization overlapped that of the membrane transport factor p115. BFA effects on Golgi structure and on MT and actin organization showed the same threshold -- all could be partially reversed after 30 min and 15 h BFA treatment but were irreversible after 40h incubation with the drug. The observed effects were not induced by signaling pathways involved in apoptotic phenomena or in ER stress response pathways. These results suggest that BFA inhibits the activity of key molecules that regulate MT and actin cytoskeleton dynamics. The findings can be used as the basis for elucidating the molecular mechanism of BFA action on the cytoskeleton.

Preparation of adsorbents applicable to pseudobiospecific ligand affinity chromatography using different spacers and ligands

Abstract In this paper, a series of different adsorbents for pseudobiospecific ligand affinity chromatography is presented. They were obtained using ligands as histidine, Cu 2+ - or Zn 2+4 -chelating iminodiacetic acid and Cibacron Bhue F3GA dye, attached to poly(butadiene-hydroxyethylmethacrylate) (PB-HEMA) matrix. The histidine and metal-iota-chelating iminodiacetic acid were attached on matrix through epichlorohydrin (ECH) or 1,4-butanediol diglycidyl ether (BDGE) as spacers and activators, while dye was directly bound onto the matrix.

Effects of experimental variables on the synthesis of porous matrices

Macroporous beads, poly(ethylene glycol dimethacrylate-co-acrylic acid) [poly(EGDMA-co-AAc)], and poly(ethylene glycol dimethacrylate-co-hydroxyethyl methacrylate) [poly(EGDMA-co-HEMA)] were prepared by the suspension polymerization technique in the presence of a porogen agent. Different experimental conditions such as amount of initiator, porogen type, and temperature were studied to optimize the polymerization systems. These hydrophilic copolymers were characterized by IR spectroscopy, scanning electron microscopy, specific surface area, and swelling in water. A new parameter, H, defined as the ratio between the equilibrium weight swelling ratio (q w ) and equilibrium volume swelling ratio (q v ), allowed to select the reaction conditions from which matrices with high capacity of water sorption and low stretching degree were reached.

Rab1b overexpression modifies Golgi size and gene expression in HeLa cells and modulates the thyrotrophin response in thyroid cells in culture

An increase in Rab1b levels induces changes in Golgi size and in gene expression. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element binding protein consensus binding. The results show a Rab1b increase in secretory cells after stimulation and suggest that this increase is required to elicit a secretory response.

Identification of a functional domain within the p115 tethering factor that is required for Golgi ribbon assembly and membrane trafficking.

Summary The tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C. elegans causes accumulation of the 170 kD soluble yolk protein (YP170) in the body cavity and retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes. Structure–function analyses of p115 have identified two homology regions (H1 and H2) within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a new C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants that lack the fourth CC domain (CC4) act in a dominant-negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi of p115 and the subsequent transfection with p115 deletion mutants, we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115. p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function, and suggest that both the CC1 and the CC4 SNARE-binding motifs participate in p115-mediated membrane tethering.

New dendronized polymers from acrylate Behera amine and their ability to produce visco-elastic structured fluids when mixed with CTAT worm-like micelles.

Two new water soluble dendronized polymers (PLn) from acrylate Behera amine monomer of different molecular weights were successfully synthesized. The polymers were characterized by FTIR, NMR, GPC and DLS. Both GPC and DLS results indicated that these PLn have a remarkable tendency to form aggregates in solution that lead to apparent molecular weights that are much higher than their theoretical values, as well as large diameters in solution. However, the addition of any PLn to water did not cause any increase in viscosity up to concentrations of 1000 ppm. The possible interactions of PLn with the cationic surfactant CTAT were explored by solution rheometry. A synergistic viscosity enhancement was found by adding small amounts of dendronized PLn polymers to a CTAT solution composed of entangled worm-like micelles. The highest association tendency with CTAT was found for PL1 at the maximum polymer concentration before phase separation (i.e., 100 ppm). The solution viscosity at low-shear rates could be increased by an order of magnitude upon addition of 100 ppm of PL1 to a 20mM CTAT solution. For this mixture, the fluid obtained was highly structured and exhibited only shear thinning behavior from the smallest shear rates employed. These PL1/CTAT mixtures exhibited an improved elastic character (as determined by dynamic rheometry) that translated in a much longer value of the cross-over relaxation time and a pronounced thixotropic behavior which are indicative of a strong intermolecular interaction. In the case of the polymer with a higher theoretical molecular weight, PL2, its association with CTAT leads to an extraordinary doubling of solution viscosity with just 0.25 ppm polymer addition to a 20mM CTAT solution. However, such synergistic viscosity enhancement saturated at rather low concentrations (25 ppm) indicating an apparent lower solubility as compared to PL1, a fact that may be related to its higher molecular weight.

Synthesis and characterization of a biospecific adsorbent containing bovine serum albumin as a ligand and its use for bilirubin retention

Epoxy-activated gels from cross-linked polybutadiene-hydroxyethylmethacrylate (PB-HEMA) copolymer and epichlorohydrin (ECH) were prepared and characterized. Albumin was covalently bonded to the matrix and used as support of affinity chromatography in bilirubin (BR) retention experiments. PB-HEMA-ECH with different amounts of immobilized albumin (between 5.20 and 6.80 mg/g dry gel) were obtained. Bilirubin retention of 3.10 mg/g of these beads was observed at 5 degrees C.

Preparation and characterization of new biospecific adsorbents with fatty acids as ligands, usable to retain bovine serum albumin

The property of albumin to bind long-chain fatty acids was exploited for the development of adsorbents containing them as ligands to isolate that protein. The synthesis of gels containing oleic or ricinoleic acids bonded to a synthetic matrix and the capacity to retain albumin are presented. The probable retention of other proteins (gelatin and lysozyme) was also tested.