Biology subjects

Kramer, D. A.

Publications and source records attributed to Kramer, D. A..

10 recordsLinked to original sources

bioRxiv2025

The CCC complex directs phagosomal maturation and bactericidal activity in macrophages through PI(3)P regulation

The evolutionarily conserved COMMD/CCDC22/CCDC93 (CCC) complex regulates endolysosomal function and is required for effective antibacterial immunity. However, the mechanisms linking the CCC complex to macrophage function remain poorly understood. Using bone marrow-derived macrophages, we found that CCC deficiency impaired bacterial clearance and disrupted phagosome maturation, as evidenced by defective phagosomal membrane remodeling, reduced phagosome-endoplasmic reticulum contacts, and impaired phagosome-lysosome fusion. These defects were independent of the CCC complex interaction partner, Retriever. CCC-deficient macrophages exhibited excessive phosphatidylinositol 3-phosphate (PI(3)P) accumulation on phagosomal membranes due to markedly reduced recruitment of the PI(3)P phosphatase MTMR2. Restoration of normal PI(3)P levels rescued bactericidal activity, establishing aberrant PI(3)P regulation as the principal defect affecting CCC-deficient macrophages. These findings identify a previously unrecognized Retriever-independent function of the CCC complex in regulating PI(3)P dynamics during phagosome maturation, a process essential for efficient bacterial clearance and innate immune defense.

cell biology↗

bioRxiv2025

The Rab32-LRMDA-Retriever Complex is a Key Regulator of Intestinal Immune Homeostasis

Maintaining intestinal homeostasis relies on the intricate interplay among the mucosal epithelium, immune system, and host microbiome. A key question is how innate immune cells sense and process microbes in the gut lumen, eliciting appropriate protective responses without causing tissue injury. Clearance of invading microbes and initiation of downstream inflammatory responses are central to this process and require proper function of the endolysosomal system. Dysfunction of this system can predispose the host to chronic inflammatory disorders and acute infections. Here, through forward genetic screening of N-ethyl-N-nitrosourea (ENU)-mutagenized mice and CRISPR/Cas9 validation, we identify Lrmda, encoding leucine-rich melanocyte differentiation-associated protein (LRMDA), as a key regulator of intestinal homeostasis. Using hematopoietic chimera and conditional knockouts, we show that LRMDA functions primarily in CD11c+ cells, including mucosal dendritic cells (DCs) and macrophages, but not in non-hematopoietic cells. Proteomic, cellular, and biochemical analyses reveal that LRMDA directly and cooperatively interacts with the endolysosome-specific small GTPase Rab32 and the endosomal recycling complex Retriever. Loss of LRMDA or Retriever function increases susceptibility to dextran sodium sulfate (DSS)-induced colitis and impairs clearance of Listeria monocytogenes. Together, our findings establish the Rab32-LRMDA-Retriever complex as a critical regulator of endolysosomal trafficking in innate immune cells, essential for maintaining intestinal immune homeostasis.

immunology↗

bioRxiv2025

Toxoplasma effector TgWIP hijacks dendritic cell actin and motility via Nck1/Grb2 and the WAVE complex

The intracellular parasite Toxoplasma gondii enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), Toxoplasma induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein Toxoplasma WAVE complex-interacting protein (TgWIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins Non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and Growth factor receptor-bound protein 2 (Grb2) as direct TgWIP interactors. TgWIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates TgWIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that TgWIP directly interacts with the actin nucleation promoting factor WAVE Regulatory Complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that TgWIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that Toxoplasma exploits to enhance host cell migration and dissemination.

microbiology↗

bioRxiv2024

Light-guided actin polymerization drives directed motility in protocells

Motility is a hallmark of lifes dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, building a minimal platform to test this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 {micro}m/min, within the range of adherent mammalian cells. Notably, our findings reveal the requirements of both branched and linear actin networks for efficient membrane protrusions. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

synthetic biology↗

bioRxiv2024

Structural basis for Retriever-SNX17 assembly and endosomal sorting

During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we report the structure of the Retriever-SNX17 complex determined using cryogenic electron microscopy (cryo-EM). Our structure reveals that the C-terminal tail of SNX17 engages with a highly conserved interface between the VPS35L and VPS26C subunits of Retriever. Through comprehensive biochemical, cellular, and proteomic analyses, we demonstrate that disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargos and altering the composition of the plasma membrane proteome. Intriguingly, we find that the SNX17-binding pocket on Retriever can be utilized by other ligands that share a consensus acidic C-terminal tail motif. By showing how SNX17 is linked to Retriever, our findings uncover a fundamental mechanism underlying endosomal trafficking of critical cargo proteins and reveal a mechanism by which Retriever can engage with other regulatory factors.

biochemistry↗

bioRxiv2023

Fibronectin: a natural barrier against prion infection

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix (ECM), mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich ECM while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues. Author summaryPrion diseases are complex fatal neurodegenerative disorders caused by a misfolded form of the cellular protein PrPC (PrPSc). Due to the potential zoonotic transmission of these disorders through animal-based food intake, it is crucial to identify the tissues in which PrPSc can accumulate and what might influence its tropism. Animal muscle (or meat) and related food products are highly consumed, raising concern of the involvement of muscle cells in prion replication. Muscle tissue from prion-affected animals contains low levels of infectivity and PrPSc is mostly associated with nerve structures rather than myofibers, whereas C2C12 myotubes, a muscle-derived cell type, efficiently replicate prions in vitro and generate high levels of infectivity compared with other cell cultures. We demonstrate a fibronectin-mediated interference with prion infection in differentiated C2C12 cultures that correlates with the findings in tissues from naturally scrapie-infected animals. Our results suggest that extracellular matrix composition, specifically regarding the presence of fibronectin, might determine prion tropism and dissemination.

biochemistry↗

bioRxiv2023

Structural Organization of the Retriever-CCC Endosomal Recycling Complex

The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of VPS35L, VPS26C and VPS29, together with the CCC complex comprising CCDC22, CCDC93, and COMMD proteins, plays a crucial role in this process. The precise mechanisms underlying Retriever assembly and its interaction with CCC have remained elusive. Here, we present the first high-resolution structure of Retriever determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog, Retromer. By combining AlphaFold predictions and biochemical, cellular, and proteomic analyses, we further elucidate the structural organization of the entire Retriever-CCC complex and uncover how cancer-associated mutations disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with Retriever-CCC-mediated endosomal recycling.

cell biology↗

bioRxiv2022

A human DCC variant causing mirror movement disorder reveals an essential role for the Wave regulatory complex in Netrin/DCC signaling

The axon guidance cue, Netrin-1, signals through its receptor DCC to attract commissural axons to the midline. Pathogenic variants in DCC frequently lead to congenital mirror movements (CMM), but how these variants impact DCC function is largely unknown. Screening of DCC in individuals with CMM recently revealed a novel variant located in a conserved motif in the cytoplasmic tail of DCC that is predicted to bind to a central actin nucleation promoting factor, the WAVE regulatory complex (WRC). Here, we use biochemical and axon guidance assays to show that this CMM-associated DCC variant is pathogenic by disrupting the interaction between DCC and the WRC. This DCC-WRC interaction is evolutionarily conserved and is required for Netrin-1 mediated commissural axon outgrowth and guidance. Together, we identify the WRC as a pivotal component of Netrin-1/DCC signaling and further provide a molecular mechanism explaining how genetic variants in DCC may lead to CMM.

neuroscience↗

bioRxiv2022

Dendrite branching receptor HPO-30 uses two novel mechanisms to regulate actin cytoskeletal remodeling

Dendrite morphogenesis is essential for neural circuit formation, yet the molecular mechanisms underlying complex dendrite branching remain elusive. Previous studies on the highly branched C. elegans PVD sensory neuron identified a membrane co-receptor complex that links extracellular signals to intracellular actin remodeling machinery, promoting high-order dendrite branching. In this complex, the claudin-like transmembrane protein HPO-30 recruits the WAVE regulatory complex (WRC) to dendrite branching sites, stimulating the Arp2/3 complex to polymerize actin. We report here our biochemical and structural analysis of this interaction, revealing that the intracellular domain (ICD) of HPO-30 is intrinsically disordered and employs two distinct mechanisms to regulate the actin cytoskeleton. First, HPO-30 ICD binding to the WRC requires dimerization and involves the entire ICD sequence, rather than a short linear peptide motif. This interaction enhances WRC activation by the GTPase Rac1. Second, HPO-30 ICD directly binds to the sides and barbed end of actin filaments. Binding to the barbed end requires ICD dimerization and inhibits both actin polymerization and depolymerization, resembling the actin capping protein CapZ. These dual functions provide an intriguing model of how membrane proteins can integrate distinct mechanisms to fine-tune local actin dynamics.

biochemistry↗

bioRxiv2022

Arf GTPase activates the WAVE Regulatory Complex through a novel binding site

Crosstalk between Rho- and Arf-family GTPases plays an important role in linking actin cytoskeletal remodeling to membrane protrusion, organelle structure, and vesicle trafficking. The central actin regulator, WAVE Regulatory Complex (WRC), is a converging point of Rac1 (a Rho-family GTPase) and Arf signaling in many processes, but how Arf promotes WRC activation is unknown. Here we reconstituted a direct interaction between Arf and WRC. This interaction can be greatly enhanced by Rac1 binding to the D site of the WRC. Arf1 binds to a newly identified conserved surface on Sra1 located between the D site and the WH2 helix of WAVE1, which can drive WRC activation using a mechanism distinct from that of Rac1. Mutating Arf binding site abolishes Arf1-WRC interaction, disrupts Arf1-mediated WRC activation, and impairs lamellipodia morphology. This work uncovers a new mechanism underlying WRC activation and provides a mechanistic foundation for studying how WRC-mediated actin polymerization links Arf and Rac signaling in the cell.

biochemistry↗