Biology subjects

Gan, L.

Publications and source records attributed to Gan, L..

8 recordsLinked to original sources

bioRxiv2018

Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

The trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. Tau secretion from neurons is the first step in tau transmission, but little is known about the cellular mechanism. Here, we report that p300/CBP, the lysine acetyltransferase that acetylates tau and regulates its homeostasis and toxicity, serves as a key regulator of tau secretion by inhibiting the autophagy-lysosomal pathway (ALP). Increased p300/CBP activity was associated with impaired function of this pathway in a tau transgenic mouse model. p300/CBP hyperactivation increased tau secretion by blocking autophagic flux. Conversely, inhibiting p300/CBP genetically or pharmacologically promoted autophagic flux, and reduced tau accumulation, tau secretion, and tau propagation in fibril-induced tau spreading models in vitro and in vivo. Our findings show that p300/CBP-induced impairment in the ALP underlies excessive unconventional secretion and pathogenic spread of tau.

neuroscience↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain

Microglia are resident immune cells that play critical roles in maintaining normal physiology of the central nervous system. Remarkably, microglia have intrinsic capacity to replenish after being acutely ablated. However, the underlying mechanisms that drive such microglial restoration remain elusive. Here, we removed microglia via CSF1R inhibitor PLX5622 and characterized repopulation both spatially and temporally. We also investigated the cellular origin of repopulated microglia and report that microglia are replenished via self-renewal, with little contribution from non-microglial lineage progenitors, including nestin+ progenitors and the circulating myeloid population. Interestingly, spatial analyses with multi-color labeling reveal that newborn microglia recolonize the parenchyma by forming distinctive clusters that maintain stable territorial boundaries over time, indicating proximal expansion nature of adult microgliogenesis and stability of microglia tiling. Temporal transcriptome profiling from newborn microglia at different repopulation stages revealed that the adult newborn microglia gradually regain steady-state maturity from an immature state that is reminiscent of neonatal stage and follow a series of maturation programs that include NF-{kappa}B activation, interferon immune activation and apoptosis, etc. Importantly, we show that the restoration of microglial homeostatic density requires NF-{kappa}B signaling as well as apoptotic egress of excessive cells. In summary, our study reports key events that take place from microgliogenesis to homeostasis re-establishment.

neuroscience↗

bioRxiv2018

The in situ structures of mono-, di-, and tri-nucleosomes in human heterochromatin

The in situ 3-D organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography (cryo-ET) to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

cell biology↗

bioRxiv2018

A particle-filter framework for robust cryoEM 3D reconstruction

Electron cryo-microscopy (cryoEM) is now a powerful tool in determining atomic structures of biological macromolecules under nearly natural conditions. The major task of single-particle cryoEM is to estimate a set of parameters for each input particle image to reconstruct the three-dimensional structure of the macromolecules. As future large-scale applications require increasingly higher resolution and automation, robust high-dimensional parameter estimation algorithms need to be developed in the presence of various image qualities. In this paper, we introduced a particle-filter algorithm for cryoEM, which was a sequential Monte Carlo method for robust and fast high-dimensional parameter estimation. The cryoEM parameter estimation problem was described by a probability density function of the estimated parameters. The particle filter uses a set of random and weighted support points to represent such a probability density function. The statistical properties of the support points not only enhance the parameter estimation with self-adaptive accuracy but also provide the belief of estimated parameters, which is essential for the reconstruction phase. The implementation of these features showed strong tolerance to bad particles and enabled robust defocus refinement, demonstrated by the remarkable resolution improvement at the atomic level.

biophysics↗

bioRxiv2018

A multi-scale model of the yeast chromosome-segregation system

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome-segregation machinery at molecular resolution in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer-kinetochore assemblies.\n\nDam1C/DASH only contacts the flat surface of the microtubule and does so with its flexible \"bridges\". In metaphase, 40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study supports a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

cell biology↗

bioRxiv2017

Cryo-ET reveals nucleosome reorganization in condensed mitotic chromosomes in vivo

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription, but the details remain unclear. Here, we use cryo-electron tomography to determine the 3-D arrangement of nucleosomes and other large nuclear features in frozen-hydrated fission-yeast cells. Nucleosomes can form irregular clusters in both interphase and mitotic cells, but they are smaller than expected for Hi-C domains. The nucleosomes are co-mingled with two features: nucleosome-free pockets and megadalton-sized \"megacomplexes\". Compared to interphase, the nucleosomes in mitotic chromosomes pack into slightly larger clusters. However, nearest-neighbor distance analysis reveals that mitotic nucleosome clusters have the same internal packing density as in interphase. Furthermore, mitotic chromosomes contain fewer megacomplexes. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: most genes are repressed but a subset is upregulated.

cell biology↗

bioRxiv2017

Natural Chromatin Is Heterogeneous And Self Associates In Vitro

The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely found in chromatin within nuclei. To determine how chromatin high-order structure is controlled, we used cryo-ET to study the undigested natural chromatin released from cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is compacted into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, some of which include face-to-face interactions. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains compact with looser nucleosome packing, even after treatment with histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore sensitive to the local environment, but generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

cell biology↗