Instituto de Histología y Embriología de Mendoza

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) 1The abbreviations used are: MVBs, multivesicular bodies; MON, monensin; Tf, transferrin; TfR, Tf receptor; AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 2-APB, 2-aminoethoxy-diphenylborate; N-Rh-Pe, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; PBS, phosphate-buffered saline; PM, plasma membrane, AchE, acetylcholinesterase; TG, thapsigargin; IP3, inositol 1,4,5-triphosphate. are endocytic organelles that contain small internal vesicles generated from inward budding of the limiting membrane. In antigen-presenting cells, the fusion of these MVBs with the plasma membrane leads to the release of internal vesicles into the extracellular space (1Clayton A. Court J. Navabi H. Adams M. Mason M.D. Hobot J.A. Newman G.R. Jasani B. J. Immunol. Methods. 2001; 247: 163-174Crossref PubMed Scopus (425) Google Scholar). The released vesicles, termed exosomes (for a review see Refs. 2Stoorvogel W. Kleijmeer M.J. Geuze H.J. Raposo G. Traffic. 2002; 3: 321-330Crossref PubMed Scopus (659) Google Scholar and 3Théry C. Zitvogel L. Amigorena S. Nat. Immunol. 2002; 2: 569-579Crossref Scopus (3835) Google Scholar), were initially described in reticulocyte maturation, where their function was to discard plasma membrane proteins that were no longer necessary, such as the transferrin receptor (4Johnstone R.M. Mathew A. Mason A.B. Teng K. J. Cell. Physiol. 1991; 147: 27-33Crossref PubMed Scopus (214) Google Scholar, 5Harding C. Heuser J. Stahl P. J. Cell Biol. 1983; 97: 329-339Crossref PubMed Scopus (1140) Google Scholar, 6Harding C. Heuser J. Stahl P. Eur. J. Cell Biol. 1984; 35: 256-263PubMed Google Scholar). Although other plasma membrane proteins (e.g. acetylcholinesterase) are secreted via exosomes, these small vesicles are devoid of both cytosolic proteins and proteins associated with other intracellular organelles, indicating that only a select group of macromolecules is shed via this pathway. Exosomes are also secreted by other cell types such as activated platelets, which may function in signaling/adhesion, thus having a role at sites of vascular injury (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar, 8Denzer K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). Exosomes from cytotoxic T cells and B lymphocytes may be involved in targeting molecules for cell death (9Peters P.J. Geuze H.J. Van der Donk H.A. Slot J.W. Griffith J.M. Stam N.J. Clevers H.C. Borst J. Eur. J. Immunol. 1989; 19: 1469-1475Crossref PubMed Scopus (190) Google Scholar) or antigen presentation (10Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2517) Google Scholar, 11Zitvogel L. Regnault A. Lozier A. Wolfers J. Flament C. Tenza D. Ricciardi-Castagnoli P. Raposo G. Amigorena S. Nat. Med. 1998; 4: 594-600Crossref PubMed Scopus (1704) Google Scholar). Despite the diverse extracellular functions that are carried out by exosomes, very little is known about the molecular machinery involved in either the formation of the MVBs or in the exosome secretory process. We have recently shown that in K562 cells, a human erythroleukemia cell line, overexpression of Rab11 regulates the exosome pathway (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Interestingly, treatment of green fluorescent protein-Rab11-transfected cells with the ionophore monensin (MON) generated large MVBs decorated with Rab11 and labeled with a fluorescent lipid that accumulates in exosomes. MON, a membrane-permeable Na+ ionophore that mediates an antiporter activity exchanging Na+ ions with H+ ions (13Pressman B.C. Ann. Rev. Biochem. 1976; 45: 501-530Crossref PubMed Scopus (1457) Google Scholar), acts on acidic intracellular organelles such as endosomes and lysosomes, causing swelling of these vesicles. MON is also known to induce Ca2+ entry by reversed activity of the Na+/Ca2+ exchanger (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). A rise in intracellular Ca2+ concentration, a universal intracellular signal (for a review see Refs. 17Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (304) Google Scholar and 18Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (678) Google Scholar), is necessary to induce regulated secretion in most cell types (reviewed in Refs.19Gerber S.H. Sudhof T.C. Diabetes. 2002; 51: S3-S11Crossref PubMed Google Scholar and 20Wasle B. Edwardson J.M. Cell. Signal. 2002; 14: 191-197Crossref PubMed Scopus (49) Google Scholar). During regulated exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane in a tightly controlled Ca2+-triggered reaction. In endocrine cells, secretory granules contain large amounts of Ca2+ ions, and it has been suggested that the high intragranular Ca2+ concentration is needed to sustain optimal exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because MON generates large MVBs in K562 cells, the aim of the present study was to MON exosome release and Ca2+ is involved in this process. Our results indicate that both MON treatment and a rise in intracellular Ca2+ markedly exosome secretion. Furthermore, the MON-stimulated exosome release was a Ca2+-dependent process. Interestingly, we have also observed that MON the of Ca2+ in the MVBs, suggesting that Ca2+ might be involved in the secretory To a physiological signal might the Ca2+-dependent exosome cells were with transferrin Our results indicate that Tf stimulates exosome release in a Ca2+-dependent cell and were from ester and were from Fluo3-AM, and were from N-(lissamine rhodamine B was from and were from and were from other were from or Cell a human erythroleukemia cell line, was in with and Exosome were from of K562 The were on at for to the cells, and at for to the Exosomes were from the by at for The exosome was in a large of and in of of of released exosomes was by the activity of an that is to these vesicles (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). activity was a described J. 16: PubMed Scopus Google Scholar). of the exosome were in of and with and in a of The was carried out in at and the in at was The the activity at of an exosomes were by the of the by of the were in for at on and to an membrane. The were for in and and with with or with were with and The were an enhanced and by MVBs with the and for fluorescent was into the plasma membrane as described J. M. G. D. Eur. J. Cell Biol. Google Scholar). an of the in was and in was with a into The was to the cells, which were for at this the was and the cells were with to the of and labeled cells were for as described and with were on and by In the cells were with by for at labeling with the fluorescent lipid. were observed these cells were an with the to and to were with a and the were with a and with the of were in the presence of for at were to the extracellular and in cells were from were in the of MON or by MON were in a the of MVBs and Exosome cells are human cells that exosomes R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar), the small internal vesicles released into the extracellular by fusion of MVBs with the plasma membrane has been shown by that treatment of K562 cells with the ionophore MON the formation of MVBs J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar, A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). the by which these large MVBs are formed and the effect of MON on exosome release have been To into these MVBs in K562 cells were labeled with the fluorescent lipid and have that this lipid is via and to the accumulates in exosomes that are secreted into the extracellular (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar, M. P. D. J. Cell Sci. PubMed Google Scholar). The lipid was to the at and cells were and at for in the or the presence of shown in MON treatment caused the formation of large MVBs labeled by the fluorescent lipid. In a of the MVBs formed is with the internal vesicles labeled with the fluorescent lipid Because fusion of the MVBs with the results in the release of exosomes, we the effect of MON on the release of exosomes from K562 cells. Exosomes are in proteins such as the transferrin receptor and R.M. A. Teng K. Blood. 1989; PubMed Google Scholar). exosomes were in the by the activity of the of and was determined by as described (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Exosomes were from the extracellular with of MON and by the of the proteins and by shown in MON a increase in exosome release in a A increase was observed by in the the activity of which was at MON in cell were observed as by for which a concentration of MON was used in the of the this concentration, cells were also for by the with of was observed In the of exosomes released was also by the fluorescent lipid this lipid accumulates in intracellular vesicles that are secreted into the extracellular as exosomes. MON also the release of exosomes labeled with the fluorescent lipid the results indicate that MON only generates large MVBs also the secretion of the internal vesicles termed exosomes. A in the Exosome has been shown that MON, a Na+ increase cytosolic Ca2+ by the Na+/Ca2+ (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). to in our the enhanced exosome release by MON was to an increase in intracellular Ca2+, we MON the intracellular Ca2+ concentration in K562 cells. this cells were for at with the intracellular Ca2+ concentration was by for of the of that was an Ca2+ and a rise in intracellular Ca2+ that was the The Ca2+ rise was by the of the intracellular Ca2+ chelator Interestingly, in the presence of the extracellular Ca2+ chelator MON the which was to Ca2+ release from intracellular no increase was indicating that the is a of Ca2+ from the extracellular The results that the increase in exosome release might be to a Ca2+-dependent To this we the MON effect on exosome release also be by Ca2+ To the Ca2+ present in the extracellular cells were for in the presence of these the Ca2+ concentration was as with the was used to intracellular Ca2+, this is a membrane-permeable that Ca2+. The released exosomes were from the and by activity as shown in both and the release of exosomes. the increase was completely by the Ca2+ and no were observed both were The that Ca2+ from the extracellular and also from intracellular is for the exosome secretion. Ca2+ in exosome release was the Ca2+ ionophore shown in with the Ca2+ ionophore stimulated exosome secretion to a as were observed both were the secretory effect of the Ca2+ ionophore was by the or is known that MON acts on acidic by the vesicle in a Ca2+ into the K. J. J. J. Cell Sci. 2001; 115: Google Scholar). We the effect of known to the of the and the R. T. A. 2001; PubMed Scopus Google Scholar). has shown that these may also intracellular Ca2+ from acidic Biochem. 1998; PubMed Scopus Google Scholar, R. Cell Calcium. 2001; PubMed Scopus Google Scholar). We have that intracellular Ca2+ in a to MON shown in stimulated the release of exosomes, to a were observed by the of with also the release of exosomes and were by extracellular Ca2+ with the chelator indicating that these via a a by indicate the of intracellular Ca2+ S. S. J. R. Biochem. J. 1996; PubMed Scopus Google for a review see Refs. A. A. Cell Calcium. 1996; 19: PubMed Scopus Google Scholar and R. 2001; PubMed Scopus Google is a that accumulates in the where cytosolic the the fluorescent membrane it has been shown used at of this is of also in intracellular and be used as an for intracellular Ca2+ A. P. B. Physiol. Rev. 1999; PubMed Scopus Google Scholar). were with for at to the MVBs were labeled with the fluorescent lipid as and the cells were with the for at shown in the large MVBs by MON treatment were labeled by Fluo3-AM, indicating that Ca2+ accumulates in these intracellular Ca2+ was also present in the large MVBs formed by The presence of the MVBs calcium in both the of the MVBs was markedly indicating that a is involved in the of the MVBs formed by MON or by and a in the of is that a of the present in the of the P.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), by a Ca2+ from other Ca2+ as as from the extracellular leads to a and increase in the concentration of treatment of K562 with this stimulated exosome secretion in a as MON, and this effect was also by a role for Ca2+ in the exosome secretory pathway and the of a Ca2+ in the process. Because stimulated exosome we were in the large MVBs by MON were also formed by treatment with shown in generated large MVBs the formation of the structures that were with that an increase in cytosolic Ca2+ is by to the MVBs, to exosome secretion. The a role in the of Ca2+ from intracellular (for a review see Refs. Cell. Full Text PDF PubMed Scopus Google Scholar and T. 2001; PubMed Scopus Google Scholar). The for the inositol a of Ca2+ for the of intracellular Ca2+ The increase in the of in the of Ca2+ present in the and the release of Ca2+ into the A. 1991; 51: PubMed Scopus Google Scholar). To this of might be involved in the exosome cells were with 2-APB, a membrane-permeable of Ca2+ that exosome results were with a of indicating that a Ca2+ rise by the of an receptor is critical for the MON-stimulated exosome the formation of the large MVBs by MON was completely by was a in the of the MVBs with the vesicles generated by MON in the of in the cells was the of the to vesicles that the release of Ca2+ via the Ca2+ at in to the of the The of the MVBs with by results indicate that Ca2+ is for of the MVBs, these structures are formed in the presence of Ca2+ to in the the of the large MVBs was only by that of Ca2+ is involved in the Ca2+ rise and the of Ca2+ in the is known that the by MON cytosolic Ca2+ by the Na+/Ca2+ the activity of a Na+/Ca2+ exchanger to be critical for the MON we an of the and Na+/Ca2+ shown in the exosome release and also completely the MON-stimulated secretion of exosomes. the formation of the MVBs generated by was completely by In the was by an of a Ca2+ results are with the that of a Na+/Ca2+ exchanger is a for the Ca2+ rise in the that leads to exosome secretion and the formation of the MVBs with Ca2+ generated by Exosome in a the results indicate that Ca2+ is a in the exosome release process. we were in a physiological stimulus might also exosome secretion in a Ca2+-dependent K562 is a human erythroleukemia cell line that high of TfR, it has been shown that of Tf to receptor intracellular Ca2+ concentration J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf might be a for exosome secretion. we Tf intracellular Ca2+ in K562 cells. this cells were with as described and human Tf was to the described for other cell types J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf an increase in Ca2+ that was for the We Tf to the exosome secretion. were for in the presence of human Tf, and exosomes were from the as described shown in Tf stimulated exosome an effect that was by or indicating that was a Ca2+-dependent process. Furthermore, the exosome release was also by 2-APB, suggesting that Ca2+ in this process. is in with a that the of to cells the of C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In this study we have shown that the ionophore MON induces the formation of large MVBs and stimulates the release of the internal vesicles exosomes. has been shown that MON induces secretion from cells (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, A. H. Biochem. 35: PubMed Scopus Google Scholar). The release of regulated secretory granules is known to be we present that the MON-stimulated exosome secretion in K562 cells is a The application of MON generated a of Ca2+ that was on both an extracellular and intracellular Ca2+ The role for Ca2+ on exosome release was by the of the Ca2+ ionophore results were with known to the of such as and an of the stimulated exosome release via a Ca2+-dependent the effect was by We have that intracellular Ca2+ in a as are in with indicating that a Ca2+ release in the Biochem. 1998; PubMed Scopus Google Scholar). it has been shown that cytosolic Ca2+ by intracellular Ca2+ in cells R. Cell Calcium. 2001; PubMed Scopus Google Scholar). our results indicate that Ca2+ is a in the exosome release process. We that this is a on their exosomes play in physiological K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). activated release exosomes at sites of vascular injury where may have a function (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar). cells also exosomes that molecules as for our that Ca2+ regulates exosome release that a signal is involved in the of cells to release these small vesicles at the The of MON to that Ca2+ an role in exosome secretion. has been that MON acts on acidic intracellular organelles such as endosomes and lysosomes, exchanging H+ for Na+ and causing swelling of these vesicles by Cell. 1983; Full Text PDF PubMed Scopus Google Scholar). it be that a might be involved in the of the large MVBs formed MON our results indicate that the formation of the endosomes is a this was completely by the Ca2+ chelator is to that the formation of these endosomes is only to swelling of the vesicles also the of from other implying fusion is that intracellular on Ca2+ A. Cell Biol. 1999; PubMed Scopus Google Scholar) it is that Ca2+ might be for the fusion involved in the of the are to this MON, as a Na+ a of Na+ the cells of H+ to the extracellular The increase in intracellular Na+ the Na+/Ca2+ exchanger in a to an increase in cytosolic Ca2+ X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). Our are in with a requirement for an of the were completely by an of the and Na+/Ca2+ Our are also with the that Na+ entry by MON the of IP3, which in releases Ca2+ from intracellular the of these might the of Ca2+ at the plasma membrane that induces a Ca2+ rise with the requirement for extracellular Ca2+ in our in this indicate that are involved in MON-stimulated exosome indicating that a Ca2+ rise by the of is critical for this the formation of the MVBs was only and completely by of the suggesting that an might be involved in the of these large Interestingly, we have observed that the large MVBs generated by are with Ca2+. is that the MON-stimulated activity might be at the plasma membrane, it is also that a effect on intracellular it has been shown that MON induces the secretion of by the membrane C. J. Biol. 1991; PubMed Scopus Google Scholar). and K. J. J. J. Cell Sci. 2001; 115: Google Scholar) have that contain high concentration of Ca2+ and function as an intracellular Ca2+ have shown that changes in in the of Ca2+ out of into the via calcium or Our results indicate a Ca2+ is involved in the release of exosomes, of this either the formation of the MVBs or the of Ca2+ the large it is that of Ca2+ present in intracellular might be for the a Ca2+ present in A. G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to the plasma membrane Ca2+ is A Ca2+ in the of J. Physiol. Cell Physiol. 2000; PubMed Google Scholar) with from the and the Ca2+ has also been secretory pathway to be a to the and is to be for the function of the secretory pathway. Interestingly, we have recently shown that MVBs in K562 cells are at in by membrane from (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Furthermore, it has been shown that endocytic vesicles from a Ca2+ that Ca2+ into the lumen of the vesicles M. B. J. J. Biochem. Mol. Biol. 35: Google Scholar). it be that a Ca2+ is involved in the formation of the MVBs and the of the Ca2+. possibility is that the Ca2+ present the MVBs is a critical role in the exosome secretory process. A role for Ca2+ in secretory and intracellular fusion has been Fusion of endosomes to the release of Ca2+ for fusion to S. M.J. J. Cell Sci. 2001; PubMed Google Scholar). in cells it has been shown that Ca2+ from granules exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the presence of on and secretory granules has been suggesting that these organelles a Ca2+ the secretory H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). we it is known that MON the the release of Ca2+. it is that the release of Ca2+ from the MVBs at the may play an role in the fusion our results indicate that Ca2+ is a critical in the MON-stimulated exosome are to the Ca2+ in this process. we have indicating that Tf intracellular calcium in K562 cells and stimulated exosome release in a Ca2+-dependent manner, suggesting that the secretion of exosomes is physiological Our results are with that the of transferrin to receptor intracellular Ca2+ and stimulates receptor in cells J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar). transferrin was stimulated by Ca2+ in cells Traffic. 2002; 3: PubMed Scopus (13) Google Scholar). Our results that a of the machinery involved in exosome secretion is regulated by Ca2+. such as and have been in where function as Ca2+ Biochem. J. 2002; PubMed Google Scholar). Tf, by increasing intracellular Ca2+, may critical of the Interestingly, in a it has been shown that the of to receptor via a the of and proteins known to be involved in C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In the to function as a signal that only also via the exosome and Ca2+ is of the of this pathway by Tf is to that exosomes released to from cells are the of the R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar). have been in K. J. PubMed Scopus Google Scholar). the physiological role of the J. J. 2002; PubMed Scopus Google Scholar) has been our that Tf stimulates exosome release as a the of TfR, to that this is a to the of We Amigorena and for critical of this We also for

and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.

During maturation, reticulocytes lose some membrane proteins that are not required on the mature red cell surface. The proteins are released into the extracellular medium associated with vesicles that are formed by budding of the endosomal membrane into the lumen of the compartment; this process results in the formation of multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. K562 cells release exosomes with similar characteristics to reticulocyte exosomes, in particular the transferrin receptor (TfR) is found associated with the vesicles. Interestingly, this cell line has been shown to possess high amounts of Rab11 compared with other Rab proteins. To assess the regulation of transferrin receptor release via exosome secretion by Rab11 in this cell type, K562 cells were stably transfected with GFP-Rab11wt or the GTP- and GDP-locked mutants. The distribution of the proteins was assessed by fluorescence microscopy. Transferrin recycling and the number of TfRs present on the surface of the transfected cells were reduced by overexpression of either Rab11wt or the mutants. The amount of released exosomes was analyzed by measuring different molecular markers present on these vesicles either biochemically or by western blot. Overexpression of the dominant-negative mutant Rab11S25N inhibited exosome release, whereas the secretion of exosomes was slightly stimulated in cells transfected with Rab11wt. Taken together, the results demonstrate that in K562 cells Rab11 modulates the exosome pathway although the exact step involved is still not known.

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

Autophagy is a major protein turnover pathway by which cellular components are delivered into the lysosomes for degradation and recycling. This intracellular process is able to maintain cellular homeostasis under stress conditions, and its dysregulation could lead to the development of physiological alterations. The autophagic activity has been found to decrease with age, likely contributing to the accumulation of damaged macromolecules and organelles during aging. Interestingly, failure of the autophagic process has been reported to worsen aging-associated diseases, such as neurodegeneration or cancer, among others. Likewise, it has been proposed in different organisms that maintenance of a proper autophagic activity contributes to extending longevity. In this review, we discuss recent papers showing the impact of autophagy on cell activity and age-associated diseases, highlighting the relevance of this process to the hallmarks of aging. Thus, understanding how autophagy plays an important role in aging opens new avenues for the discovery of biochemical and pharmacological targets and the development of novel anti-aging therapeutic approaches.

Pathogens evolved mechanisms to invade host cells and to multiply in the cytosol or in compositionally and functionally customized membrane-bound compartments. Coxiella burnetii, the agent of Q fever in man is a Gram-negative gamma-proteobacterium which multiplies in large, acidified, hydrolase-rich and fusogenic vacuoles with phagolysosomal-like characteristics. We reported previously that C. burnetii phase II replicative compartments are labelled by LC3, a protein specifically localized to autophagic vesicles. We show here that autophagy in Chinese hamster ovary cells, induced by amino acid deprivation prior to infection with Coxiella increased the number of infected cells, the size of the vacuoles, and their bacterial load. Furthermore, overexpression of GFP-LC3 or of GFP-Rab24 - a protein also localized to autophagic vacuoles - likewise accelerated the development of Coxiella-vacuoles at early times after infection. However, overexpression of mutants of those proteins that cannot be targeted to autophagosomes dramatically decreased the number and size of the vacuoles in the first hours of infection, although by 48 h the infection was similar to that of non-transfected controls. Overall, the results suggest that transit through the autophagic pathway increases the infection with Coxiella by providing a niche more favourable to their initial survival and multiplication.

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.

Galectins are a family of endogenous glycan-binding proteins that have crucial roles in a broad range of physiological and pathological processes. As a group, these proteins use both extracellular and intracellular mechanisms as well as glycan-dependent and independent pathways to reprogramme the fate and function of numerous cell types. Given their multifunctional roles in both tissue fibrosis and cancer, galectins have been identified as potential therapeutic targets for these disorders. Here, we focus on the therapeutic relevance of galectins, particularly galectin 1 (GAL1), GAL3 and GAL9 to tumour progression and fibrotic diseases. We consider an array of galectin-targeted strategies, including small-molecule carbohydrate inhibitors, natural polysaccharides and their derivatives, peptides, peptidomimetics and biological agents (notably, neutralizing monoclonal antibodies and truncated galectins) and discuss their mechanisms of action, selectivity and therapeutic potential in preclinical models of fibrosis and cancer. We also review the results of clinical trials that aim to evaluate the efficacy of galectin inhibitors in patients with idiopathic pulmonary fibrosis, nonalcoholic steatohepatitis and cancer. The rapid pace of glycobiology research, combined with the acute need for drugs to alleviate fibrotic inflammation and overcome resistance to anticancer therapies, will accelerate the translation of anti-galectin therapeutics into clinical practice.

The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment. Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine, a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5-/- cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis, the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.