Reconstitution of intracellular clathrin-coated vesicle formation

Clathrin-coated vesicle (CCV) formation is a major membrane remodelling process important for membrane traffic in eukaryotic cells. CCVs are formed at the plasma membrane during endocytosis, and at early/recycling endosomes and the trans-Golgi network during intracellular traffic. The plasma membrane is easier to access experi- mentally in vivo from the cell exterior, while intracellular compartments lack this ad- vantage. To overcome this barrier and to study intracellular CCV dynamics and func- tional requirements, we have reconstituted intracellular CCV formation on-demand, using minimal machinery. The clathrin-binding region of the b2 subunit of AP-2 was used as a `hook' which can be attached inducibly to an `anchor' protein on a membrane surface. Rerouting the hook to an anchor by chemical dimerisation was sufficient to form CCVs at mitochondria, ER, Golgi and lysosomes. As mitochondria are not part of canonical membrane trafficking, I investigated synthetic clathrin-coated pit forma- tion on mitochondria in detail. CCPs on the mitochondria (termed mitoPits), form within minutes after induction. Electron microscopy and live cell imaging revealed that initiation, maturation and scission steps of CCV formation were faithfully recon- stituted. MitoPits are double membraned invaginations that tend to form on surfaces with higher curvature. These observations suggests that enough force is generated by our synthetic system to deform both the inner and outer mitochondrial membranes and for budding of the mitoPits. Vesicle budding was shown not to depend on any scission molecule tested (dynamin, Drp1, Vps4a, actin), suggesting that intracellular CCVs do not need a scission factor. To conclude, unlike endocytosis, clathrin-coating may be sufficient for intracellular CCV budding. Given the differences in phospholipid profiles of mitochondrial membranes and plasma membrane, the phospholipid composition of the membrane may have a negligible role in CCV formation

Pattern engineering of living bacterial colonies using meniscus-driven fluidic channels

Creating adaptive, sustainable, and dynamic biomaterials is a forthcoming mission of synthetic biology. Engineering spatially organized bacterial communities has a potential to develop such bio-metamaterials. However, generating living patterns with precision, robustness, and a low technical barrier remains as a challenge. Here we present an easily implementable technique for patterning live bacterial populations using a controlled meniscus-driven fluidics system, named as MeniFluidics. We demonstrate multiscale patterning of biofilm colonies and swarms with submillimeter resolution. Utilizing the faster bacterial spreading in liquid channels, MeniFluidics allows controlled bacterial colonies both in space and time to organize fluorescently labeled Bacillus subtilis strains into a converged pattern and to form dynamic vortex patterns in confined bacterial swarms. The robustness, accuracy, and low technical barrier of MeniFluidics offer a tool for advancing and inventing new living materials that can be combined with genetically engineered systems, and adding to fundamental research into ecological, evolutional, and physical interactions between microbes

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment ‘hook’ to an ‘anchor’ protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation – initiation, invagination, and even fission – were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation

Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs