AAK1 and BMP2K are serine/threonine kinases traditionally known for phosphorylating AP2 during clathrin-mediated endocytosis (CME), but their broader roles remained incompletely defined. Here, using motif-guided in silico, biochemical, and phosphoproteomic screens, we identify PDLIM5 and Talin1 as direct AAK1/BMP2K substrates. Despite high kinase-domain similarity, only AAK1 promotes cell migration and potentiates focal adhesion (FA) turnover. Live-cell imaging shows that AAK1 recruitment to FAs peaks as disassembly begins. The conserved AAK1 C-terminal PDZ-binding motif mediates direct, low-affinity binding to PDLIM5, providing a plausible mechanism for localized substrate access. Dynamic analyses of phospho-mimetic and phospho-null mutants support a model in which AAK1-dependent phosphorylation promotes timely release of PDLIM5 and Talin1 during FA disassembly. These findings reveal a kinase-driven contribution to FA turnover distinct from protease- and phosphatase-based mechanisms and suggest that functional divergence between AAK1 and BMP2K may provide a strategy to modulate cell migration with reduced impact on CME.

Clathrin-mediated endocytosis (CME), the main mechanism for swift, selective protein uptake in eukaryotic cells, initiates with adaptor protein AP2 recruitment to the plasma membrane (PM). AP2 recognizes PM-associated PtdIns(4,5)P2 and protein cargo for internalization. Nonetheless, many aspects of this process remain unclear due to their in vivo complexity. Here, a thermodynamic and time-resolved structural analysis of AP2 binding to different biomimetic PM was undertaken under physiological conditions using a combination of neutron reflectometry, interfacial tensiometry and rheology, and atomic force microscopy. The resultant in vitro data replicated previous in vivo observations, as well as yielded biophysical insights into normal and aborted CME. The presence of cargo may not be pivotal for the "activating" conformational change of AP2. However, the presence of cargo extends AP2's residence time on the membrane surface, due to slower on- and off-rates, thereby tentatively giving sufficient time for CME to proceed fully. Moreover, upon interaction with AP2, phospholipid lateral diffusion decreases markedly, inducing a gel phase attributed to creating a percolated network involving AP2 on the membrane, which could potentially serve as a mechanism for modulating subsequent clathrin binding. The subsequent clathrin polymerization at the membrane is dependent on the AP2's clathrin binding sequence.

Clathrin assembly lymphoid myeloid leukaemia protein (CALM) is involved in the formation of clathrin-mediated endocytic coats by virtue of binding many proteins involved in the process, including clathrin itself and AP2 cargo adaptor complex. CALM is able to specifically recognize the inner leaflet of the plasma membrane by binding the membrane’s phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Here, a quantitative biophysical approach —combining neutron/X-ray scattering, solid-state NMR, atomic force microscopy, and quartz crystal microbalance with dissipation monitoring—, was exploited to investigate CALM interaction with PtdIns(4,5)P2-presenting model membranes. The presented experimental data reveal CALM’s folded domain partially embeds (12 To selectively internalize proteins into the endosomal system, eukaryotic cells rely on clathrin adaptors such as the clathrin assembly lymphoid myeloid leukaemia protein (CALM), but details regarding the underlying mechanism remain unexplored. Here, the authors characterize the effect of CALM on lipid packaging density and membrane remodelling through a combination of in vitro studies, neutron reflectometry, grazing incidence X-ray diffraction, atomic force microscopy and solid-state nuclear magnetic resonance, and show that CALM’s amphiphilic N-terminal helix embeds into the membrane to stabilize protein docking.

In this issue of Structure, Horatscheck et al.1 report the isolation and characterization of two improved small molecule drugs for the selective inhibition of clathrin-mediated endocytosis.

The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28. We term this approach PACTAC (PCSK9-Antibody Clearance-Targeting Chimeras).

After primary infection, human cytomegalovirus (HCMV) establishes lifelong persistence, underpinned by latent carriage of the virus with spontaneous reactivation events. In the immune-competent, primary infection or reactivation from latency rarely causes disease. However, HCMV can cause significant disease in immune-compromised individuals such as immune-suppressed transplant patients. Latency, where the viral genome is carried in the absence of the production of infectious virions, can be established in undifferentiated cells of the myeloid lineage. A number of stimuli can cause virus reactivation from latency to occur, beginning with the induction of viral immediate-early (IE) lytic gene expression. The suppression of viral IE gene expression to establish and maintain latent infection is known to result from a balance of viral and cellular factors. One key viral factor involved in this is the G protein-coupled receptor US28. Recently, we have shown that US28 is targeted for degradation by a modified nanobody (PCTD-Vun100bv) based on the novel PACTAC (PCSK9-antibody clearance-targeting chimeras) approach for targeted protein degradation. Furthermore, we have shown that this PCTD-Vun100bv-induced degradation of US28 results in IE gene expression in experimentally latently infected CD14+ monocytes. However, HCMV also establishes latency in CD34+ bone marrow cells, the progenitors of CD14+ cells. Here, we show that PCTD-Vun100bv also causes US28 degradation in these CD34+ primary cells, again resulting in the induction of viral IE gene expression. Additionally, we show that PCTD-Vun100bv can target US28 in naturally latently infected CD14+ monocytes from an HCMV-seropositive donor, allowing these latently infected cells to be killed by HCMV-specific cytotoxic T cells from that same donor. These observations support the view that targeting US28 for degradation during natural latency could be a tractable ‘shock-and-kill’ strategy to target the latent HCMV reservoir in myeloid cells.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.125 character summary FCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s### Competing Interest StatementThe authors have declared no competing interest.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence–structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5) P 2 -containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.