SPECIAL ISSUE: Reconstituting Cell Biology
CELL SCIENTISTS TO WATCH
CELL SCIENCE AT A GLANCE
Summary: Up-to-date cell biology tools that enable deconstruction of the complex environments through which cells move in tissues are demonstrated via a hand-drawn poster.
Summary: This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology, and how it will continue to do so as researchers reconstitute increasingly complex biomolecular processes.
Summary: Understanding of the mechanisms involved in T cell receptor activation has been greatly advanced by reconstitution systems. Here, we review how clustering of the T cell receptor impacts on signal transduction in T cells and how reconstitution systems have contributed to this knowledge.
Summary: This Review focuses on the ongoing efforts to recapitulate the formation of a tubular ER network using purified proteins such as the curvature-stabilizing proteins and the fusion GTPases.
Summary: Autophagy is a versatile recycling system that ensures turnover of cytoplasmic material. This Review highlights in vitro reconstitution studies that helped to discover fundamental mechanisms of the pathway.
The depolymerase activity of MCAK shows a graded response to Aurora B kinase phosphorylation through allosteric regulation
Summary: The kinesin-13 MCAK has a compact conformation in solution but is extended when bound to microtubules. Aurora B phosphorylation of MCAK inhibits depolymerase activity by disrupting its extended conformation.
Claudin-4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins
Summary: Successful reconstitution of the tight junction protein claudin-4 drove interface formation between giant unilamellar vesicles. These interfaces were sufficient to act as molecular fences toward proteins but not lipids.
Summary: Drusen, a hallmark of age-related macular degeneration, are produced by three-dimensional spheroids of retinal pigment epithelial cells. This spheroid culture can be used as a new tool to investigate drusen biogenesis.
Membrane fluctuations and acidosis regulate cooperative binding of ‘marker of self’ protein CD47 with the macrophage checkpoint receptor SIRPα
Summary: Binding of membrane proteins smooths the fluctuating membrane and facilitates more binding, which can be very important for weak immunological interactions such as the interaction of macrophage SIRPα with CD47 on cancer cells.
Force-history dependence and cyclic mechanical reinforcement of actin filaments at the single molecular level
Summary: Actin subunit interactions are reinforced by cyclic force loading, providing insight into force regulation in actin cytoskeletal dynamics.
Summary: Epithelia growing under tubular confinement are shown to detach and constrict to form a narrow tube in a process that depends on curvature of the substrate, cell shape and contractility.
Summary: Experiments combined with simulations show that myosin-2 motors drive actin network contraction via a polarity-sorting mechanism that relies on dwelling of myosins at the plus end of actin filaments.
Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex
Summary: Myosin-II-driven disassembly, when counterbalanced by Arp2/3-dependent actin polymerization, generates continuous actin network turnover in a biomimetic minimal cortex.
Summary: In microtubule gliding motility assays, gliding velocity increases with dynein surface density and MT length. We hypothesize that this effect is related to a mechanical activation of individual dynein motors.
Summary: PLK4 binds to microtubules and self-assembles into condensates that recruit tubulin and trigger de novo microtubule-organising centre formation in vitro.
Spatial positioning of EB family proteins at microtubule tips involves distinct nucleotide-dependent binding properties
Summary: In vitro reconstitution of tip tracking with EB1, EB2 and EB3 shows that these three proteins sense the nucleotide state of both β-tubulins flanking their binding site.
Summary: Target membrane integrity during influenza hemagglutinin-mediated lipid mixing between viral and target membrane is dependent upon target membrane spontaneous curvature.
Summary: We present a new methodology to reconstitute proteins on membranes with complex geometry by means of vesicle fusion, unveiling a putative novel function of CHMP2B as a diffusion barrier in dendritic spines.
Drp1 polymerization stabilizes curved tubular membranes similar to those of constricted mitochondria
Summary: Drp1 follows an unprecedented molecular mechanism when assembling on lipid tubes, which sets it apart from other dynamins. Drp1 stabilizes mitochondrial curvature rather than being a membrane scissor.
The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor
Summary: In vivo and in vitro experiments demonstrate Arf-GDP regulation of GBF1 recruitment to a heat-labile and protease-sensitive site on Golgi membranes. This recruitment requires the HDS1 and HDS2 domains.
TOOLS AND RESOURCES
Summary: Decellularised matrices derived from discarded animal-house material provide a physiologically relevant 3D-platform for studying cancer cell behaviour that is easy to use, inexpensive and embraces the 3Rs.
A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly
Summary: This article describes the development of artificial nuclear membranes as a powerful and simplistic synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly
Regulated reconstitution of spindle checkpoint arrest and silencing through chemically induced dimerisation in vivo
Summary: Abscisic acid is used as a chemically induced dimeriser (CID) to bring together Mph1 kinase and Spc7 and initiate spindle checkpoint signalling in vivo. Simply washing out the abscisic acid enables checkpoint silencing to be studied.
Summary: Immune cell–cell interactions are reconstituted in free-standing vesicles, allowing the spatiotemporal aspects of immune synapse formation to be investigated.
Summary: A new in vitro assay for measuring the growth and dynamics of single microtubules within the complexity of total budding yeast soluble protein.
ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes
Summary: This paper outlines a simple protocol to reconstitute integrin chimeras within liposomes and use flow cytometry to quantify the impact of lipid composition on integrin–talin interactions.