An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.

Dynamic association of the ULK1 complex with omegasomes during autophagy induction.

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities.

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination.

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

Optimized immunofluorescence staining protocol for imaging germinal centers in secondary lymphoid tissues of vaccinated mice.

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

ATG13 dynamics in nonselective autophagy and mitophagy: insights from live imaging studies and mathematical modeling.

During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy.Abbreviations: ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED: Discosoma red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.

CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+.

The versatility of Ca2+ as a second messenger lies in the complex manner in which Ca2+ signals are generated. How information contained within the Ca2+ code is interpreted underlies cell function. Recently, we identified CAPRI and RASAL as related Ca2+-triggered Ras GTPase-activating proteins. RASAL tracks agonist-stimulated Ca2+ oscillations by repetitively associating with the plasma membrane, yet CAPRI displays a long-lasting Ca2+-triggered translocation that is refractory to cytosolic Ca2+ oscillations. CAPRI behavior is Ca2+- and C2 domain-dependent but sustained recruitment is predominantly Ca2+ independent, necessitating integration of Ca2+ by the C2 domains with agonist-evoked plasma membrane interaction sites for the pleckstrin homology domain. Using an assay to monitor Ras activity in real time, we correlate the spatial and temporal translocation of CAPRI with the deactivation of H-Ras. CAPRI seems to low-pass filter the Ca2+ signal, converting different intensities of stimulation into different durations of Ras activity in contrast to the preservation of Ca2+ frequency information by RASAL, suggesting sophisticated modes of Ca2+-regulated Ras deactivation.

Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies.

Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies.

Autophagosome Biogenesis Machinery.

We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well coordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of nonselective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform.

The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.

Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles.

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

Control of Ras cycling by Ca2+.

Ras GTPases are binary switches, cycling between an inactive GDP-bound form and an active GTP-bound form at the membrane. They transduce signals into the cytoplasm via effector pathways that regulate cell growth, differentiation and apoptosis. Ras activation is enhanced by guanine nucleotide exchange factors (GEFs); deactivation is accelerated by GTPase-activating proteins (GAPs). Recently, new roles for Ca(2+) and diacylglycerol (DAG) in the control of Ras cycling have emerged with the discovery of a series of novel GEFs and GAPs. These regulators of Ras cycling are likely to play a key role in the information processing of Ca(2+) and DAG signals.

P-Rex2, a new guanine-nucleotide exchange factor for Rac.

We have identified a new guanine-nucleotide exchange factor, P-Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P-Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)-trisphosphate and the beta gamma subunits of heterotrimeric G proteins in vitro and in vivo. P-Rex2 has structure, activity and regulatory properties similar to P-Rex1 but has divergent tissue distribution, as P-Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein-coupled receptors and phosphoinositide 3-kinase.

Characteristics and requirements of basal autophagy in HEK 293 cells.

Basal autophagy-here defined as macroautophagic activity during cellular growth in normal medium containing amino acids and serum-appears to be highly active in many cell types and in animal tissues. Here we characterized this pathway in mammalian HEK 293 cells. First, we examined, side by side, three compounds that are widely used to reveal basal autophagy by blocking maturation of autophagosomes: bafilomycin A 1 (BafA1), chloroquine and vinblastine. Only BafA1 appeared to be without complicating side effects. Chloroquine partially inhibited mechanistic target of rapamycin (MTOR) activity, which would induce autophagy induction as well as block autophagosome maturation. Vinblastine caused the distribution of early omegasome components into punctate phagophore assembly sites, and therefore it would also induce autophagy, complicating interpretation. Basal autophagy was significantly sensitive to inhibition by wortmannin, and therefore required formation of phosphatidylinositol 3-phosphate (PtdIns3P), but it was twice as resistant to wortmannin as starvation-induced autophagy. We also determined that basal autophagy was significantly suppressed by MTOR activation brought about by overexpression of RHEB or activated RAGs. Finally we investigated the spatial relationship of nascent autophagosomes to the endoplasmic reticulum (ER) or to mitochondria by live imaging experiments under conditions that reveal basal autophagy (with BafA1 treatment), or upon MTOR inactivation (which would result in autophagy induction). Side-by-side comparison showed that under both basal and induced autophagy, 100% of autophagosomes first appeared in close proximity to ER strands. In parallel measurements, 40% were in close proximity to mitochondria under both conditions. We concluded that in HEK 293 cells, basal autophagy is mechanistically similar to that induced by MTOR inactivation in all aspects examined.

Live cell imaging of early autophagy events: omegasomes and beyond.

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer. Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution. Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation-contraction coupling and inotropic stimulation.

Narrow, tubular, inward projections of the sarcolemma ('T-tubules') are an established feature of adult mammalian ventricular myocytes that enables them to generate the whole-cell Ca2+ transients and produce coordinated contraction. Loss of T-tubules can occur during ageing and under pathological conditions, leading to altered cardiac excitation-contraction coupling. In contrast to adult ventricular cells, atrial myocytes do not generally express an extensive T-tubule system at any stage of development, and therefore rely on Ca2+ channels around their periphery for the induction of Ca2+ signalling and excitation-contraction coupling. Consequently, the characteristics of systolic Ca2+ signals in adult ventricular and atrial myocytes are temporally and spatially distinct. However, although atrial myocytes do not have the same regularly spaced convoluted T-tubule structures as adult ventricular cells, it has been suggested that a proportion of adult atrial cells have a more rudimentary tubule system. We examined the structure and function of these atrial tubules, and explored their impact on the initiation and recovery of Ca2+ signalling in electrically paced myocytes. The atrial responses were compared to those in adult ventricular cells that had intact T-tubules, or that had been chemically detubulated. We found that tubular structures were present in a significant minority of adult atrial myocytes, and were unlike the T-tubules in adult ventricular cells. In those cells where they were present, the atrial tubules significantly altered the on-set, amplitude, homogeneity and recovery of Ca2+ transients. The properties of adult atrial myocyte Ca2+ signals were different from those in adult ventricular cells, whether intact or detubulated. Excitation-contraction coupling in detubulated adult ventricular myocytes, therefore, does not approximate to atrial signalling, even though Ca2+ signals are initiated in the periphery of the cells in both of these situations. Furthermore, inotropic responses to endothelin-1 were entirely dependent on T-tubules in adult ventricular myocytes, but not in atrial cells. Our data reveal that that the T-tubules in atrial cells impart significant functional properties, but loss of these tubular membranes does not affect Ca2+ signalling as dramatically as detubulation in ventricular myocytes.