AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody.

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.

The Rho GTPase Rif signals through IRTKS, Eps8 and WAVE2 to generate dorsal membrane ruffles and filopodia.

Rif induces dorsal filopodia but the signaling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS (also known as BAIAP2L1) through its I-BAR domain. Rif also interacts with Pinkbar (also known as BAIAP2L2) in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant-negative Rif inhibits the formation of IRTKS-induced morphological structures, and Rif activity is blocked in IRTKS-knockout (KO) cells. To further define the Rif-IRTKS signaling pathway, we identify Eps8 and WAVE2 (also known as WASF2) as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS signaling, whereas WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic Escherichia coli infection, might compete for Rif for interaction with the I-BAR domain of IRTKS. Based on this evidence, we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53 (also known as BAIAP2), IRTKS and Pinkbar, as a central mechanism to modulate cell morphology.

Talin determines the nanoscale architecture of focal adhesions.

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.

mDia1 and WAVE2 proteins interact directly with IRSp53 in filopodia and are involved in filopodium formation.

Filopodia are dynamic actin-rich cell surface protrusions involved in cell migration, axon guidance, and wound healing. The RhoGTPase Cdc42 generates filopodia via IRSp53, a multidomain protein that links the processes of plasma membrane deformation and actin dynamics required for their formation in mammalian cells. The Src homology 3 domain of IRSp53 binds to the actin regulators Mena, Eps8, WAVE1, WAVE2, mDia1, and mDia2. We show that mDia1 and WAVE2 synergize with IRSp53 to form filopodia. IRSp53 also interacts directly with these two proteins within filopodia, as observed in acceptor photobleaching FRET studies. Measurement of filopodium formation by time-lapse imaging of live cells also revealed that depleting neuronal cells of either mDia1 or WAVE2 protein decreases the ability of IRSp53 to induce filopodia. In contrast, IRSp53 does not appear to partner WAVE1 or mDia2 to give rise to these structures. In addition, although all three isoforms of mDia are capable of inducing filopodia, IRSp53 requires only mDia1 to do so. These findings suggest that mDia1 and WAVE2 are important Src homology 3 domain partners of IRSp53 in forming filopodia.

Nanoscale imaging by superresolution fluorescence microscopy and its emerging applications in biomedical research.

Superresolution microscopy, an ensemble of light microscopy methods developed with the aim of surpassing the resolution limit imposed by diffraction, has been at the forefront of imaging technology innovations in recent years. By harnessing advances in fluorophore photophysics, fluorescent protein engineering, optics, and image processing, rapid strides have been made in enhancing imaging resolution via 3 major approaches: structured illumination microscopy, stimulated emission depletion microscopy, and single-molecule localization microscopy. From a diffraction-limited resolution of ~250 nm, an improvement of more than an order of magnitude down to ~10 nm can now be attained, converging upon the size scale of the macromolecular building blocks of cells. This opens up the possibility of direct visualization of molecular-scale architecture and interactions of specific proteins in biological structures that are important to health and disease. Here, theoretical foundations and practical considerations of superresolution microscopy in 2- and 3-dimensional imaging are discussed, along with their recent applications in addressing biological questions.

Mitochondrial Dysfunction in CD4+ T Effector Memory RA+ Cells.

Human ageing is accompanied by poor responses to infection and decreased vaccine efficacy. While the causes of this can be attributed to defects in the immune system that increase with age, it is unknown whether mitochondrial dysfunction may also contribute to these phenomena. This study aims to assess mitochondrial dysfunction in CD4+ terminal effector memory T cells re-expressing CD45RA (TEMRA) cells and other CD4+ memory T cell subtypes, which are increased in number in the elderly population, with respect to how their metabolic responses to stimulation are altered compared to CD4+ naïve T cells. In this study, we show that CD4+ TEMRA cells exhibit altered mitochondrial dynamics compared to CD4+ naïve cells and CD4+ central and effector memory cells, with a 25% reduction in OPA1 expression. CD4+ TEMRA and memory cells show increased upregulation of Glucose transporter 1 following stimulation and higher levels of mitochondrial mass compared to CD4+ naïve T cells. Additionally, TEMRA cells exhibit a decrease in mitochondrial membrane potential compared to other CD4+ memory cell subsets by up to 50%. By comparing young to aged individuals, more significant mitochondria mass and lower membrane potential were observed in CD4+ TEMRA of young individuals. In conclusion, we suggest that CD4+ TEMRA cells may be impaired with respect to their metabolic response to stimulation, possibly contributing to impaired responses to infection and vaccination.

I-BAR domains, IRSp53 and filopodium formation.

Filopodia and lamellipodia are dynamic actin-based structures that determine cell shape and migration. Filopodia are thought to sense the environment and direct processes such as axon guidance and neurite outgrowth. Cdc42 is a small GTP-binding protein and member of the RhoGTPase family. Cdc42 and its effector IRSp53 (insulin receptor phosphotyrosine 53 kDa substrate) have been shown to be strong inducers of filopodium formation. IRSp53 consists of an I-BAR (inverse-Bin-Amphiphysin-Rvs) domain, a Cdc42-binding domain and an SH3 domain. The I-BAR domain of IRSp53 induces membrane tubulation of vesicles and dynamic membrane protrusions lacking actin in cells. The IRSp53 SH3 domain interacts with proteins that regulate actin filament formation e.g. Mena, N-WASP, mDia1 and Eps8. In this review we suggest that the mechanism for Cdc42-driven filopodium formation involves coupling I-BAR domain-induced membrane protrusion with SH3 domain-mediated actin dynamics through IRSp53.

Rif-mDia1 interaction is involved in filopodium formation independent of Cdc42 and Rac effectors.

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.

Combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP.

BACKGROUND: Lamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53-/- mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins. METHODS: We performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology. RESULTS: In HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized. CONCLUSIONS: Our data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8.

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

pS421 huntingtin modulates mitochondrial phenotypes and confers neuroprotection in an HD hiPSC model.

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by mutant huntingtin (mHTT). Phosphorylation at serine-421 (pS421) of mHTT has been shown to be neuroprotective in cellular and rodent models. However, the genetic context of these models differs from that of HD patients. Here we employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, we generated isogenic hiPSC lines in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phospho-resistant alanine (S421A). We observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although we observed no effect of S421D on mHTT clearance or axonal transport, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, our analysis revealed modulation of several aspects of mitochondrial form and function. These include mitochondrial surface area, volume, and counts, as well as improved mitochondrial membrane potential and oxidative phosphorylation. Our study validates the protective role of pS421 on mHTT and highlights a facet of the relationship between mHTT and mitochondrial changes in the context of human physiology with potential relevance to the pathogenesis of HD.

Exploiting the protein corona around gold nanorods for low-dose combined photothermal and photodynamic therapy.

A nanodevice comprising human serum (HS) protein corona coated gold nanorods (NRs) has been developed to perform both photothermal therapy (PTT) and photodynamic therapy (PDT) simultaneously at a very low dose under irradiation by a single laser. Here, we exploit the protein corona to load a photosensitizer, chlorin e6 (Ce6), to form NR-HS-Ce6, whose excitation wavelength matches with the longitudinal surface plasmon resonance (LSPR) of NRs. When excited by a single laser, the NRs caused photothermal ablation of cancer cells while Ce6 simultaneously produced reactive oxygen species (ROS) to kill cancer cells through oxidative stress in PDT. We found that the protein corona did not affect the photothermal heating of NRs and observed more than 5-fold increase in ROS generation when Ce6 was loaded on NR-HS compared to free HS-Ce6 dissolved in HS. The uptake of Ce6 by Cal 27 oral squamous cell carcinoma (OSCC) cells also increased 57-fold when loaded on NR-HS compared to free HS-Ce6. While both PDT and PTT have established modest success in reducing cancer cell viability on their own, we have shown that the combined therapy can achieve near complete eradication (95.2% cell kill) of cancer cells even at an extremely low dose of 50 pM of NR-HS-Ce6 containing an equivalent of 7.67 µg mL-1 Au and 4.83 nM Ce6. This near complete cell kill at such a low dose has not been reported previously. The advantages of this nanoscale delivery system showcase the application of protein corona in cancer treatment instead of considering it as an undesirable biological artefact.

Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.

Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250 nm laterally and >500 nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20 nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750 nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.

mDia1-3 in mammalian filopodia.

mDia proteins are members of the formin family of actin nucleating proteins that polymerize linear actin filaments. Such filaments form the core of thin, tubular, membrane-bound cell surface protrusions known as filopodia, which are a major feature of mammalian cell morphology. Filopodia are dynamic structures that help cells sense environmental cues, and play a role in cell migration, axon guidance, angiogenesis and other processes. RhoGTPases bind to and control the activity of mDia proteins, and several other binding partners of the three mDia1 isoforms-mDia1, mDia2 and mDia3-have been documented. Two independent pathways controlling mammalian filopodium formation have emerged, with one driven by the RhoGTPase Cdc42, and the other by Rif. While mDia2 has been the main formin implicated in forming filopodia, mDia1 has recently surfaced as the key formin utilized by both the Cdc42 and Rif pathways to drive filopodial protrusion.

Rho GTPase Cdc42 is a direct interacting partner of Adenomatous Polyposis Coli protein and can alter its cellular localization.

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC¹â»¹6³8 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC¹â»¹³³8 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins.

F-BAR domain proteins: Families and function.

The F-BAR domain is emerging as an important player in membrane remodeling pathways. F-BAR domain proteins couple membrane remodeling with actin dynamics associated with endocytic pathways and filopodium formation. Here, we provide a comprehensive analysis of F-BAR domain proteins in terms of their evolutionary relationships and protein function. F-BAR domain containing proteins can be categorized into five subfamilies based on their phylogeny which is consistent with the additional protein domains they possess, for example, RhoGAP domains, Cdc42 binding sites, SH3 domains and tyrosine kinase domains. We derive a protein-protein interaction network suggesting that dynamin1/2, N-WASP, Huntingtin, intersectin and Cdc42 are central nodes influencing F-BAR domain protein function.

The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics.

The Cdc42 effector IRSp53 is a strong inducer of filopodia formation and consists of an Src homology domain 3 (SH3), a potential WW-binding motif, a partial-Cdc42/Rac interacting binding region motif, and an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain. We show that IRSp53 interacts directly with neuronal Wiskott-Aldrich syndrome protein (N-WASP) via its SH3 domain and furthermore that N-WASP is required for filopodia formation as IRSp53 failed to induce filopodia formation in N-WASP knock-out (KO) fibroblasts. IRSp53-induced filopodia formation can be reconstituted in N-WASP KO fibroblasts by full-length N-WASP, by N-WASPDeltaWA (a mutant unable to activate the Arp2/3 complex), and by N-WASPH208D (a mutant unable to bind Cdc42). IRSp53 failed to induce filopodia in mammalian enabled (Mena)/VASP KO cells, and N-WASP failed to induce filopodia when IRSp53 was knocked down with RNA interference. The IRSp53 I-BAR domain alone induces dynamic membrane protrusions that lack actin and are smaller than normal filopodia ("partial-filopodia") in both wild-type N-WASP and N-WASP KO cells. We propose that IRSp53 generates filopodia by coupling membrane protrusion through its I-BAR domain with actin dynamics through SH3 domain binding partners, including N-WASP and Mena.