Matthew N.J. Seaman

Bio: Matthew N.J. Seaman is an academic researcher from University of Cambridge. The author has contributed to research in topics: Retromer & Endosome. The author has an hindex of 39, co-authored 71 publications receiving 11720 citations. Previous affiliations of Matthew N.J. Seaman include University of Texas Southwestern Medical Center & University of California, San Diego.

Induction of autophagy and inhibition of tumorigenesis by beclin 1.

Abstract: The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

Autophagy Genes Are Essential for Dauer Development and Life-Span Extension in C. elegans

Abstract: Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.

Clathrin-mediated endocytosis in AP-2–depleted cells

Abstract: We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

A Membrane Coat Complex Essential for Endosome-to-Golgi Retrograde Transport in Yeast

Abstract: We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G. Marcusson, J.-L. Cereghino, and S.D. Emr. 1997. J. Cell Biol. 137:79-92). By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p. This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p. Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction. Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles. We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding. Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation. Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation. Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.

Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

Abstract: fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called “retromer” that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

Abstract: Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.