The rockefeller university

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with micro- tubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells ' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Despite therapeutic efficacy observed with immune checkpoint blockade in advanced melanoma, many tumors do not respond to treatment, representing a need for new therapies. Here, we have generated chimeric antigen receptor (CAR) T cells targeting TYRP1, a melanoma differentiation antigen expressed on the surface of melanomas, including rare acral and uveal melanomas. TYRP1-targeted CART cells demonstrate antigen-specific activation and cytotoxic activity in vitro and in vivo against human melanomas independent of the MHC alleles and expression. In addition, the toxicity to pigmented normal tissues observed with T lymphocytes expressing TYRP1-targeted TCRs was not observed with TYRP1-targeted CAR T cells. Anti-TYRP1 CAR T cells provide a novel means to target advanced melanomas, serving as a platform for the development of similar novel therapeutic agents and as a tool to interrogate the immunobiology of melanomas.

The Harpy Eagle (Harpia harpyja) is an iconic species that inhabits forested landscapes in Neotropical regions, with decreasing population trends mainly due to habitat loss, and currently classified as vulnerable. Here, we report on a chromosome-scale genome assembly for a female individual combining long reads, optical mapping, and chromatin conformation capture reads. The final assembly spans 1.35 Gb, with N50scaffold equal to 58.1 Mb and BUSCO completeness of 99.7%. We built the first extensive transposable element (TE) library for the Accipitridae to date and identified 7,228 intact TEs. We found a burst of an unknown TE similar to 13-22 million years ago (MYA), coincident with the split of the Harpy Eagle from other Harpiinae eagles. We also report a burst of solo-LTRs and CR1 retrotransposons similar to 31-33 MYA, overlapping with the split of the ancestor to all Harpiinae from other Accipitridae subfamilies. Comparative genomics with other Accipitridae, the closely related Cathartidae and Galloanserae revealed major chromosome-level rearrangements at the basal Accipitriformes genome, in contrast to a conserved ancient genome architecture for the latter two groups. A historical demography reconstruction showed a rapid decline in effective population size over the last 20,000 years. This reference genome serves as a crucial resource for future conservation efforts towards the Harpy Eagle.

In eukaryotes, the leading strand DNA is synthesized by Pol epsilon and the lagging strand by Pol delta. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Pol epsilon catalytic domain has been determined, how Pol epsilon interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Pol epsilon-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Pol epsilon catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Pol epsilon. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding. In eukaryotes, the leading strand DNA polymerase Pol epsilon synthesises the DNA with higher processivity when in complex with the DNA clamp PCNA. Here, the authors report two cryo-EM structures of human Pol epsilon bound to the PCNA clamp and a DNA substate, revealing the conformational changes associated with incoming nucleotide binding.

Judy Lieberman is a professor of pediatrics and adjunct professor of genetics at Harvard Medical School and an endowed chair in cellular and molecular medicine. Her lab studies cytotoxic T lymphocytes (CTL), key cells in the immune defense against viral infection and cancer, as well as molecular pathways activated by the granzymes, and how RNA interference (RNAi) regulates cell differentiation in health and disease states. We spoke to Judy about advice for early career researchers, how she first become interested in cytotoxic T lymphocytes, and key people who have provided mentorship across her career.

Upon antigenic stimulation, naive CD4(+) T cells can give rise to phenotypically distinct effector T helper cells and long-lived memory T cells. We computationally reconstructed the in vivo trajectory of CD4(+) T cell differentiation during a type I inflammatory immune response and identified two distinct differentiation paths for effector and precursor central memory T cells arising directly from naive CD4(+) T cells. Unexpectedly, our studies revealed heterogeneity among naive CD4(+) T cells, which are typically considered homogeneous save for their diverse T cell receptor usage. Specifically, a previously unappreciated population of naive CD4(+) T cells sensing environmental type I IFN exhibited distinct activation thresholds, suggesting that naive CD4(+) T cell differentiation potential may be influenced by environmental cues. This population was expanded in human viral infection and type I IFN response-lined autoimmunity. Understanding the relevance of naive T cell heterogeneity to beneficial and maladaptive T cell responses may have therapeutic implications for adoptive T cell therapies in cancer immunotherapy and vaccination.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

A key step for metastatic outgrowth involves the generation of a deeply altered microenvironment (niche) that supports the malignant behavior of cancer cells. The complexity of the metastatic niche has posed a significant challenge in elucidating the underlying programs driving its origin. Here, by focusing on early stages of breast cancer metastasis to the lung in mice, we describe a cancer-dependent chromatin remodeling and activation of developmental programs in alveolar type 2 (AT2) cells within the niche. We show that metastatic cells can prime AT2 cells into a reprogrammed multilineage state. In turn, this cancer-induced reprogramming of AT2 cells promoted stem-like features in cancer cells and enhanced their initiation capacity. In conclusion, we propose the concept of "reflected stemness" as an early phenomenon during metastatic niche initiation, wherein metastatic cells reprogram the local tissue into a stem-like state that enhances intrinsic cancer-initiating potential, creating a positive feedback loop where tumorigenic programs are amplified.