Endoplasmic reticulum-plasma membrane contact sites (ER-PM CSs) are evolutionarily conserved membrane domains found in all eukaryotes, where the ER closely interfaces with the PM. This short distance is achieved in plants through the action of tether proteins such as synaptotagmins (SYTs). Arabidopsis comprises five SYT members (SYT1-SYT5), but whether they possess overlapping or distinct biological functions remains elusive. SYT1, the best-characterized member, plays an essential role in the resistance to abiotic stress. This study reveals that the functionally redundant SYT1 and SYT3 genes, but not SYT5, are involved in salt and cold stress resistance. We also show that, unlike SYT5, SYT1 and SYT3 are not required for Pseudomonas syringae resistance. Since SYT1 and SYT5 interact in vivo via their SMP domains, the distinct functions of these proteins cannot be caused by differences in their localization. Interestingly, structural phylogenetic analysis indicates that the SYT1 and SYT5 clades emerged early in the evolution of land plants. We also show that the SYT1 and SYT5 clades exhibit different structural features in their SMP and Ca2+ binding of their C2 domains, rationalizing their distinct biological roles.

BACKGROUND: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor.
METHODS: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging.
RESULTS: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction.
CONCLUSIONS: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV-C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2-25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1-3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed \"open\" and \"closed\" states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both \"open\" and \"closed\" states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1-3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

The ankyrin repeat-rich membrane spanning (ARMS), a transmembrane neuronal scaffold protein, plays a fundamental role in neuronal physiology, including neuronal development, polarity, differentiation, survival and angiogenesis, through interactions with diverse partners. Previous studies have shown that the ARMS negatively regulates brain-derived neurotrophic factor (BDNF) secretion by interacting with Synaptotagmin-4 (Syt4), thereby affecting neurogenesis and the development and function of the nervous system. However, the molecular mechanisms of the ARMS/Syt4 complex assembly remain unclear. Here, we confirmed that the ARMS directly interacts with Syt4 through its N-terminal ankyrin repeats 1-8. Unexpectedly, both the C2A and C2B domains of Syt4 are necessary for binding with the ARMS. We then combined the predicted complex structural models from AlphaFold2 with systematic biochemical analyses using point mutagenesis to underline the molecular basis of ARMS/Syt4 complex formation and to identify two conserved residues, E15 and W72, of the ARMS, as essential residues mediating the assembly of the complex. Furthermore, we showed that ARMS proteins are unable to interact with Syt1 or Syt3, indicating that the interaction between ARMS and Syt4 is specific. Taken together, the findings from this study provide biochemical details on the interaction between the ARMS and Syt4, thereby offering a biochemical basis for the further understanding of the potential mechanisms and functional implications of the ARMS/Syt4 complex formation, especially with regard to the modulation of BDNF secretion and associated neuropathies.

The recruitment of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by N-methyl-D-aspartate (NMDA) receptor-dependent postsynaptic Ca2+ influx. Synaptotagmin (Syt)-1 and -7 have been proposed as Ca2+ sensors for AMPA receptor exocytosis but are functionally redundant. Here, we identify a cytosolic C2 domain-containing Ca2+-binding protein, Copine-6, that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary neurons, which is rescued by wild-type Copine-6 but not Ca2+-binding mutants. In contrast, Copine-6 loss of function does not affect steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/Syt-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca2+-binding mutants, restores activity-dependent exocytosis of AMPA receptors in Syt-1/Syt-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca2+ sensor that mediates AMPA receptor exocytosis during synaptic potentiation.

BACKGROUND: The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.
METHODS: For this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.
RESULTS: The biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.
CONCLUSIONS: We have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Many signaling processes rely on information decoding at the plasma membrane, and membrane-associated proteins and their complexes are fundamental for regulating this process. Still many questions exist as to how protein complexes are assembled and function at membrane sites to change identity and dynamics of membrane systems. Peripheral membrane proteins containing a calcium and phospholipid-binding C2-domain can act in membrane-related signaling by providing a tethering function so that protein complexes form. C2 domain proteins termed C2-DOMAIN ABSCISIC ACID-RELATED (CAR) proteins are plant-specific, and the functional relevance of this C2 domain protein subgroup is just emerging. The ten Arabidopsis CAR proteins CAR1 to CAR10 have a single C2 domain with a plant-specific insertion, the so-called \"CAR-extra-signature\" or also termed \"sig domain\". Via this \"sig domain\" CAR proteins can bind signaling protein complexes of different kinds and act in biotic and abiotic stress, blue light and iron nutrition. Interestingly, CAR proteins can oligomerize in membrane microdomains, and their presence in the nucleus can be linked with nuclear protein regulation. This shows that CAR proteins may play unprecedented roles in coordinating environmental responses and assembling required protein complexes to transmit information cues between plasma membrane and nucleus. The aim of this review is to summarize structure-function characteristics of the CAR protein family and assemble findings from CAR protein interactions and physiological functions. From this comparative investigation we extract common principles about the molecular operations that CAR proteins may fulfill in the cell. We also deduce functional properties of the CAR protein family based on its evolution and gene expression profiles. We highlight open questions and suggest novel avenues to prove and understand the functional networks and roles played by this protein family in plants.

Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5\'-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.

Membrane proteins are broadly classified as transmembrane (TM) or peripheral, with functions that pertain to only a single bilayer at a given time. Here, we explicate a class of proteins that contain both transmembrane and peripheral domains, which we dub transmembrane membrane readers (TMMRs). Their transmembrane and peripheral elements anchor them to one bilayer and reversibly attach them to another section of bilayer, respectively, positioning them to tether and fuse membranes while recognizing signals such as phosphoinositides (PIs) and modifying lipid chemistries in proximity to their transmembrane domains. Here, we analyze full-length models from AlphaFold2 and Rosetta, as well as structures from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, using the Membrane Optimal Docking Area (MODA) program to map their membrane-binding surfaces. Eukaryotic TMMRs include phospholipid-binding C1, C2, CRAL-TRIO, FYVE, GRAM, GTPase, MATH, PDZ, PH, PX, SMP, StART and WD domains within proteins including protrudin, sorting nexins and synaptotagmins. The spike proteins of SARS-CoV-2 as well as other viruses are also TMMRs, seeing as they are anchored into the viral membrane while mediating fusion with host cell membranes. As such, TMMRs have key roles in cell biology and membrane trafficking, and include drug targets for diseases such as COVID-19.