Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Macroautophagic cargo sequestration assays

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterisation of the total secretome of this zoonotic parasite. Fasciola secretes at least two sub-populations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialised cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic datasets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular machinery required for EV biogenesis is constitutively expressed across the intra-mammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Resolución N° 17 ¿Cuales son los lineamientos que considera la Cámara de Comercio en el estudio de actas contentivas de la renuncia y aceptación del cargo de miembro de Junta por el órgano máximo de la sociedad?

Servicios registrales

Resolución No. 025 del 26 de febrero de 2014 ¿Existe un control de legalidad a cargo de la Cámara de comercio sobre la verificación o reconocimiento de la titularidad de quienes detentan acciones en una Sociedad Anónima?

La Cámara de Comercio de Bogotá inscribió el acta No. AA-021 de la asamblea de accionistas de la SOCIEDAD DE TELECOMUNICACIONES, INGENIERÍA MERCADEO TELEVISIÓN POR CABLE Y SERVICIOS SAS, en la cual se hizo nombramiento de gerente general, gerente ejecutivo y presidente de la asamblea de accionistas.

The Columbian Newspaper vol. IV no. 1105- June 12, 1813

The "Columbian" was established by Mr. Holt in 1808 as an "organ of the Clintonians" (Journalism in the United States, from 1690-1872 By Frederic Hudson) and a replacement to the "Citizen". It was a passionate Democratic paper that supported both Jefferson and Madison. Holt later sold the "Columbian" to Alden Spooner, who published the Long Island "Star". The Columbian ceased in 1821. This issue includes the following War of 1812 topics: Page 2: copies of letters from U.S. Gen. H. Dearborn describing the U.S. attack on Ft. George 27 May 1813; letter from Gen. H. Dearborn describing U.S. pursuit of British troops at Beaver Dams and the U.S. capture of Fort Erie; list of U.S. killed and wounded in Commodore Isaac Chauncey's squadron in attack on York 27 April, 1813; list of killed and wounded in Commodore Isaac Chauncey's squadron in attack on Ft. George 27 May 1813; abstract of the cartel for the exchange of prisoners of war between Great Britain and the United States; mention of troop movements through New York on their way to the battle front; list of persons killed or lost on the privateer Saratoga; Page 3: account of the loss of the U.S. war ships the Growler and Eagle to the British; news from Quebec and Kingston of troop movements; U.S. ship Syren unsuccessfully pursues British ship Herald; account of a U.S. cargo ship being captured by a British military ship; account of U.S. ship Siro capturing British ship Loyal Sam; report from Halifax of recent British troop and ship movements; list of recent troop enrollments in various states; report of British troop build up in Kingston;

Independent Chronicle Vol. XLVI No. 3470- July 14, 1814

A semi-weekly paper that was published from 1801 to 1817. It was previously called the Independent chronicle and the universal advertiser, and was later the Independent chronicle and Boston patriot (Semiweekly). Publishers were Abijah Adams and Ebenezer Rhoades. Topics of interest in this issue include: Page 1: report of Independence Day celebration including multiple toasts to the military; Page 2: report of South Carolina militia marching home; report of U.S. schooner Alligator being lost to a tornado in Port Royal; report of 10,000 militia and 2,000 regular Army troops to be used in defense of the District of Columbia and vicinity; report U.S. is to try to raise 100,000 militia for defense; news of British deserters providing information to the U.S.; report of skirmishes along the U.S. coast; statement from U.S. Navy Capt. David Porter stating he had taken possession of Sir Henry Martin's Island; report of British troops burning a militia barracks in Lewiston, N.Y.; 4 accounts of the battle of Chippewa and the taking of Fort Erie; statement from Maj. Gen. Brown in praise of his troops in the battle of Chippewa; copy of General Brown's orders to attack at Fort Erie; essay arguing against U.S. citizens boarding enemy ships; report of peace treaty rumours; Page 3: account of Capt. Porter's capture and subsequent escape from the British; list of American prisoners held on the British ship Nymph; report of British cargo ship captured by U.S. war ship; Proclamation from James Madison pardoning military deserters if they return to military service; announcement to officers and soldiers in the military offering reward for the return of military deserters; Page 4: 2 reward advertisements for return of military deserters;

Schooner Ranger document

A page of entries detailing the financial history of the Schooner Ranger, a Great Lakes cargo ship, from 1810-1815. The ship sustained damage in 1811 on its journey from Detroit to Black Rock, in which 3 people drowned. The ship was subsequently “destroyed by the enemy in January 1813”. In August 1815, the ship’s account was settled. The settlement acknowledged that the ship was “taken by the U.S. Navy Officers for the use of the Government”, and is signed by Porter, Barton & Co., and George Kibbe.

The Columbian, 12 June 1813 (vol. 4, no. 1105) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812. Issue for June 12, 1813 includes copies of letters from U.S. Gen. H. Dearborn describing the U.S. attack on Ft. George 27 May 1813; letter from Gen. H. Dearborn describing U.S. pursuit of British troops at Beaver Dams and the U.S. capture of Fort Erie; list of U.S. killed and wounded in Commodore Isaac Chauncey’s squadron in attack on York 27 April, 1813; list of killed and wounded in Commodore Isaac Chauncey’s squadron in attack on Ft. George 27 May 1813; abstract of the cartel for the exchange of prisoners of war between Great Britain and the United States; mention of troop movements through New York on their way to the battle front; list of persons killed or lost on the privateer Saratoga; account of the loss of the U.S. war ships the Growler and Eagle to the British; news from Quebec and Kingston of troop movements; report of launching of British ship Sir George Prevost; announcement of sending of 5 American infantry companies to Burlington from Bennington, Vermont; U.S. ship Syren unsuccessfully pursues British ship Herald; account of a U.S. cargo ship being captured by a British military ship; account of U.S. ship Siro capturing British ship Loyal Sam; report from Halifax of recent British troop and ship movements; list of recent troop enrollments in various states; report of British troop build up in Kingston.

Extracto que forma el Mayor General de infantería de las relaciones dadas por los cuerpos de su cargo acerca de los muertos, heridos y acciones particulares que cada uno tuvo en la función de Puente Calderón el día 7 de enero de 1811

UANL