Li-Lin Du

Bio: Li-Lin Du is an academic researcher from Tsinghua University. The author has contributed to research in topics: Schizosaccharomyces pombe & DNA repair. The author has an hindex of 29, co-authored 79 publications receiving 7517 citations. Previous affiliations of Li-Lin Du include Scripps Research Institute & Yale University.

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.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide 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.

Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast

Abstract: Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as -H2AX and -H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate -H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of -H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22 (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2 cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that -H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that -H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.

Abstract: Replication of a eukaryotic genome is a challenging task that is often made more difficult by conditions that interfere with replisome progression. These circumstances include DNA lesions that obstruct replicative polymerases, drugs that target DNA polymerases or enzymes required to synthesize deoxynucleoside triphosphates (dNTPs), and protein complexes bound to DNA (7, 36). Stalled replication forks are prone to collapse, regression, and recombination (32). Collapsed forks are among the most serious forms of DNA damage and as such pose a grave threat to cell survival and genome integrity (28). Discovering how cells cope with aberrant replication forks is therefore essential for understanding mechanisms of genome maintenance. Studies of budding and fission yeasts have uncovered a network of proteins that form the replication checkpoint (7, 36). Central to this network are protein kinases of the ATM/ATR family, such as Mec1 in the budding yeast Saccharomyces cerevisiae and Rad3 in the fission yeast Schizosaccharomyces pombe. In fission yeast, Rad3 forms a complex with Rad26 and functions together with a trimeric checkpoint clamp (Rad1-Rad9-Hus1) and five-subunit checkpoint clamp loader (Rad17-RFC2-RFC3-RFC4-RFC5) to sense stalled replication forks and transmit a checkpoint signal. These proteins act together with Mrc1, a mediator of the replication checkpoint, to activate the replication checkpoint effector Cds1, a protein kinase homologous to Rad53 in budding yeast and Chk2 in humans (1, 46). Budding yeast rad53 mutants starved of dNTPs will arrest in S phase and accumulate aberrant DNA structures such as regressed forks or hemireplicated regions, and they are unable to fully resolve replication intermediates when dNTP levels are restored (30, 44, 47). How Rad53 preserves stalled forks is unknown, but there is accumulating evidence that it and Cds1 control phosphorylation of several replication and recombination proteins (7, 36). These proteins include Mus81, a subunit of the Mus81-Eme1 DNA endonuclease complex. Mus81-Eme1 is required for recovery from collapsed forks and is thought to be a component of Holliday junction resolvase in fission yeast. Mus81 physically interacts with Cds1 homologs in budding yeast, fission yeast, and humans (5, 6, 11, 22). Mammalian Chk2 plays an important role in controlling the apoptotic response to DNA damage, apparently through its ability to control the p53 tumor suppressor, and recent evidence has indicated that Chk2 is a bona fide tumor suppressor (21, 45). In some situations, stalled forks are important for cell vitality, as is the case for cell type switching in fission yeast (12, 13). Mating type in fission yeast is determined by the expressed gene cassette at the mat1 locus. Mating type switching occurs when the DNA cassette at mat1 is replaced by one of two silent donor cassettes. Switching requires a strand-specific imprinting event that occurs when mat1 is replicated in a specific direction. The direction of replication of mat1 is determined by polar replication fork pausing and termination sites located near mat1 and is dependent on several proteins. One of these proteins is Swi1, a 971-amino acid (aa) protein that has ∼25% sequence identity to Drosophila melanogaster Timeless, mammalian Tim1, and budding yeast Tof1 (13). Timeless controls circadian rhythms in Drosophila (35), whereas mouse Tim1 is an essential nuclear protein that does not regulate circadian rhythms but whose function is unknown (19). Tof1, first identified as topoisomerase 1-associated factor in a two-hybrid screen, is involved in DNA damage responses during S phase (16). Mutant tof1 cells exhibit no obvious phenotypes and are not sensitive to genotoxic agents, but deletion of TOF1 enhances the genotoxic-sensitive phenotype of rad9 mutants. Rad9 is a mediator protein that facilitates activation of Rad53 by Mec1 (18, 42). Deletion of TOF1 exacerbated the Rad53 activation defect of rad9 mutants, suggesting that Tof1 and Rad9 act in redundant pathways to control Rad53 activation (16). Fission yeast swi1 mutants have additional phenotypes that are unconnected to mating type switching (13). They have a reduced growth rate that is exacerbated by mutations in top1, which encodes topoisomerase I. Swi1 is essential for viability in a mutant that is partially defective for DNA polymerase alpha. These studies indicated that Swi1 might have a more general role in DNA replication. Here we report that Swi1 is crucial for survival of replication fork arrest. Swi1 has both Cds1-dependent and -independent functions. Swi1 appears to act early in the response to fork arrest, perhaps as a replisome component, to stabilize forks in a configuration that is recognized by the replication checkpoint sensors.

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.

Rho GTPases in cell biology.

Abstract: Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.

Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

Abstract: The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.