Biology subjects

Lippert, A. H.

Publications and source records attributed to Lippert, A. H..

5 recordsLinked to original sources

bioRxiv2025

MYO1F interactome reveals the SH3-domain linked CASS complex at podosomes and the phagocytic cup

MYO1F, a long-tailed myosin of class I, is selectively expressed in immune cells and upregulated in microglia associated with neurodegenerative pathogenesis. The intracellular functions of myosin motors involve adaptor proteins, which regulate cargo attachment and intracellular motor recruitment. To define the MYO1F interactome, we performed an in situ proximity labelling-based proteomic analysis in human myeloid U937 cells. We identified a distinct SH3-domain-dependent adaptor module comprising CD2AP, ASAP1, SH3BP2, and SH3KBP1 (CASS complex), which localizes with MYO1F in podosomes and phagocytic cups. Structural modelling and mutagenesis confirmed multivalent proline-rich motif interactions of the CASS complex with the MYO1F SH3 domain. Further deletions revealed a second group of membrane-associated adaptor proteins that bind to the MYO1F pleckstrin homology (PH) domain. Immunofluorescence in macrophages and microglia confirmed the conserved localization of MYO1F and its adaptors at actin-rich podosomes and phagocytic cups. Functional assays demonstrated that MYO1F recruitment to the phagocytic cup requires motor activity and intact PH and SH3 domains. This study provides the first comparative interactome of MYO1F and its paralogue MYO1E and supports a role for MYO1F in podosomes and during phagocytosis in both peripheral and brain-resident myeloid cells.

cell biology↗

bioRxiv2025

Actin conformation dynamics precede force generation in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) are cells of the adaptive immune system that are able to recognise and kill cancerous or virally infected target cells. The biochemical basis of CTL-mediated killing has been examined in great detail. Recent work has underscored the importance of physical forces in T cell function too, but how CTLs sense and exert force during migration and killing is not fully understood. Here, by expressing actin conformation probes based on the CH domain of utrophin, we directly visualize regions of altered F-actin conformation in primary CTLs during migration and killing. By combining these probes with traction force microscopy, we correlate external force with the internal cytoskeleton. We show that actin conformation is regulated upstream of force production, and that force exertion at the cytotoxic immune synapse temporally follows actin conformation dynamics. Our work offers novel insight into the relationship between cellular force production and F-actin conformation in important primary immune cells.

cell biology↗

bioRxiv2020

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

The T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing soft bilayers with physiological levels of mechanical resistance (Youngs modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

biophysics↗

bioRxiv2018

Membrane potential regulates the dynamic localisation of mammalian proteasomes

Proteasomes are abundant molecular machines distributed throughout the eukaryotic cell to facilitate protein degradation. We previously showed that proteasomes can re-organize and assemble into foci bodies in response to proteotoxic stress1, which we termed transient aggregate-associated droplets (TAADs)2. Here, we use single-molecule localization microscopy to quantify re-organization of mammalian proteasomes in different subcellular regions during proteotoxic stress to engage with invading protein aggregates. We determine that [~]60% of 20S proteasomes are capped by at least one 19S particle, and this ratio remains constant during stress. Using single-particle tracking, we show that cells confine global proteasomal movement when experiencing proteotoxic stress via a cytoskeleton-dependent mechanism. Similar limitations on proteasome movement are also observed upon membrane depolarization and repolarization events by patch clamp, which directly induce proteasomes to move away or towards the plasma membrane, respectively. Together, our experiments reveal distinct modes of proteasome motion dependent on cellular requirements, and propose that proteasome transport is restricted upon aggregate invasion to facilitate their subsequent degradation.

cell biology↗

bioRxiv2018

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Of fundamental importance, they are transient structures that generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about the morphological transition and cellular dynamics during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi photon imaging of GFP-transgenic chick embryos. We specifically focussed on the dynamic changes in two principle regions within the somite (the medial and lateral domains) to investigate extensive morphological changes. Furthermore, by using quantitative analysis and cell tracking, we were able to capture for the first time a progenitor cell bulk movement towards the rostral-medial domain of the myotome, where skeletal muscle formation first initiates.

developmental biology↗