Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters.

Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.

Abnormal axonal development and severe epileptic phenotype in Dynamin-1 (DNM1) encephalopathy.

Dynamin-1 (DNM1) is involved in synaptic vesicle recycling, and DNM1 mutations can lead to developmental and epileptic encephalopathy. The neuroimaging of DNM1 encephalopathy has not been reported in detail. We describe a severe phenotype of DNM1 encephalopathy showing characteristic neuroradiological features. In addition, we reviewed previously reported cases who have DNM1 pathogenic variants with white matter abnormalities. Our case presented drug-resistant seizures from 1 month of age and epileptic spasms at 2 years of age. Brain MRI showed no progression of myelination, progression of diffuse cerebral atrophy, and a thin corpus callosum. Proton magnetic resonance spectroscopy showed a decreased N-acetylaspartate peak and diffusion tensor imaging presented with less pyramidal decussation. Whole-exome sequencing revealed a recurrent de novo heterozygous variant of DNM1. So far, more than 50 cases of DNM1 encephalopathy have been reported. Among these patients, delayed myelination occurred in two cases of GTPase-domain DNM1 encephalopathy and in six cases of middle-domain DNM1 encephalopathy. The neuroimaging findings in this case suggest inadequate axonal development. DNM1 is involved in the release of synaptic vesicles with the inhibitory transmitter GABA, suggesting that GABAergic neuron dysfunction is the mechanism of refractory epilepsy in DNM1 encephalopathy. GABA-mediated signaling mechanisms play important roles in axonal development and GABAergic neuron dysfunction may be cause of white matter abnormalities in DNM1 encephalopathy.

Reversal of cell, circuit and seizure phenotypes in a mouse model of DNM1 epileptic encephalopathy.

Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene (DNM1) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.

Clinical, Radiological, and Genetic Characterization of a Patient with a Novel Homoallelic Loss-of-Function Variant in DNM1.

Heterozygous pathogenic variants in DNM1 are linked to an autosomal dominant form of epileptic encephalopathy. Recently, homozygous loss-of-function variants in DNM1 were reported to cause an autosomal recessive form of developmental and epileptic encephalopathy in unrelated patients. Here, we investigated a singleton from a first-degree cousin marriage who presented with facial dysmorphism, global developmental delay, seizure disorder, and nystagmus. To identify the involvement of any likely genetic cause, diagnostic clinical exome sequencing was performed. Comprehensive filtering revealed a single plausible candidate variant in DNM1. Sanger sequencing of the trio, the patient, and her parents, confirmed the full segregation of the variant. The variant is a deletion leading to a premature stop codon and is predicted to cause a protein truncation. Structural modeling implicated a complete loss of function of the Dynamin 1 (DNM1). Such mutation is predicted to impair the nucleotide binding, dimer formation, and GTPase activity of DNM1. Our study expands the phenotypic spectrum of pathogenic homozygous loss-of-function variants in DNM1.

Reduced dynamin-1 levels in neurons lacking MUNC18-1.

MUNC18-1 (also known as syntaxin-binding protein-1, encoded by Stxbp1) binds to syntaxin-1. Together, these proteins regulate synaptic vesicle exocytosis and have a separate role in neuronal viability. In Stxbp1 null mutant neurons, syntaxin-1 protein levels are reduced by 70%. Here, we show that dynamin-1 protein levels are reduced at least to the same extent, and transcript levels of Dnm1 (which encodes dynamin-1) are reduced by 50% in Stxbp1 null mutant brain. Several, but not all, other endocytic proteins were also found to be reduced, but to a lesser extent. The reduced dynamin-1 expression was not observed in SNAP25 null mutants or in double-null mutants of MUNC13-1 and -2 (also known as Unc13a and Unc13b, respectively), in which synaptic vesicle exocytosis is also blocked. Co-immunoprecipitation experiments demonstrated that dynamin-1 and MUNC18-1 do not bind directly. Furthermore, MUNC18-1 levels were unaltered in neurons lacking all three dynamin paralogues. Finally, overexpression of dynamin-1 was not sufficient to rescue neuronal viability in Stxbp1 null mutant neurons; thus, the reduction in dynamin-1 is not the single cause of neurodegeneration of these neurons. The reduction in levels of dynamin-1 protein and mRNA, as well as of other endocytosis proteins, in Stxbp1 null mutant neurons suggests that MUNC18-1 directly or indirectly controls expression of other presynaptic genes.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model.

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Dephospho-dynamin 1 coupled to activity-dependent bulk endocytosis participates in epileptic seizure in primary hippocampal neurons.

BACKGROUND AND PURPOSE: Epilepsy is a severe neurological and mental disorder, and not all patients adequately respond to the current treatments. Dynamin 1 plays a key role in synaptic endocytosis and the modulation of neurological function. MATERIAL AND METHODS: Cultured hippocampal neurons were used in the study. First, the viability of neurons was determined by the CCK-8 assay after culturing in magnesium-free medium, DMSO, dynasore (dynamin agonist), and PIP2 (dynamin antagonist). Then, the effect of dynasore on seizure activity was evaluated. Next, we tested the levels of phospho-dynamin 1/total dynamin 1 and dynamin 1 mRNA in the control group and four epilepsy groups. Moreover, the uptake of tetramethylrhodamine-dextran in the different groups was measured. RESULTS: Dephospho-dynamin 1 expression was significantly increased in hyperexcitable neurons, while there was no change in total dynamin 1 level. The level of dephospho-dynamin 1 in hyperexcitable neurons was reduced when cultured with dynasore but increased with PIP2 treatment. Activity-dependent bulk endocytosis (ADBE) was upregulated in hyperexcitable neurons. Along with a decrease in dephospho-dynamin 1 level, ADBE was also downregulated with dynasore treatment, while PIP2 did not affect ABDE. The close link between the dephosphorylation status of dynamin 1 and ADBE suggests that ADBE activation depends on dynamin 1 dephosphorylation. CONCLUSION: Dephospho-dynamin 1 triggers ADBE to meet the requirements of high-frequency discharges during epileptic seizures.

Dynamin 1 controls vesicle size and endocytosis at hippocampal synapses.

Following calcium-triggered vesicle exocytosis, endocytosis regenerates vesicles to maintain exocytosis and thus synaptic transmission, which underlies neuronal circuit activities. Although most molecules involved in endocytosis have been identified, it remains rather poorly understood how endocytic machinery regulates vesicle size. Vesicle size, together with the transmitter concentration inside the vesicle, determines the amount of transmitter the vesicle can release, the quantal size, that may control the strength of synaptic transmission. Here, we report that, surprisingly, knockout of the GTPase dynamin 1, the most abundant brain dynamin isoform known to catalyze fission of the membrane pit's neck (the last step of endocytosis), not only significantly slowed endocytosis but also increased the synaptic vesicle diameter by as much as ∼40-64% at cultured hippocampal synapses. Furthermore, dynamin 1 knockout increased the size of membrane pits, the precursor for endocytic vesicle formation. These results suggest an important function of dynamin other than its well-known fission function - control of vesicle size at the pit formation stage.

Genomic and Phenotypic Characterization of Experimentally Selected Resistant Leishmania donovani Reveals a Role for Dynamin-1-Like Protein in the Mechanism of Resistance to a Novel Antileishmanial Compound.

The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

Loss-of-function variants in DNM1 cause a specific form of developmental and epileptic encephalopathy only in biallelic state.

BACKGROUND: Developmental and epileptic encephalopathies (DEEs) represent a group of severe neurological disorders characterised by an onset of refractory seizures during infancy or early childhood accompanied by psychomotor developmental delay or regression. DEEs are genetically heterogeneous with, to date, more than 80 different genetic subtypes including DEE31 caused by heterozygous missense variants in DNM1. METHODS: We performed a detailed clinical characterisation of two unrelated patients with DEE and used whole-exome sequencing to identify causative variants in these individuals. The identified variants were tested for cosegregation in the respective families. RESULTS: We excluded pathogenic variants in known, DEE-associated genes. We identified homozygous nonsense variants, c.97C>T; p.(Gln33*) in family 1 and c.850C>T; p.(Gln284*) in family 2, in the DNM1 gene, indicating that biallelic, loss-of-function pathogenic variants in DNM1 cause DEE. CONCLUSION: Our finding that homozygous, loss-of-function variants in DNM1 cause DEE expands the spectrum of pathogenic variants in DNM1. All parents who were heterozygous carriers of the identified loss-of-function variants were healthy and did not show any clinical symptoms, indicating that the type of mutation in DNM1 determines the pattern of inheritance.

DNM1: A Prognostic Biomarker Associated with Immune Infiltration in Colon Cancer-A Study Based on TCGA Database.

AIM: The aim of our work was to determine the utility of DNM1 as a biomarker for the diagnosis and prognosis of colon cancer (CC). METHODS: DNM1 expression variations in CC vs. normal tissues were investigated using The Cancer Genome Atlas (TCGA) database. The association of DNM1 expression levels with the clinicopathological variables in CC prognosis was investigated using logistic regression analyses. Independent prognostic factors for CC were evaluated using univariate and multivariate Cox regression analyses. The correlation between DNM1 expression and immune cell infiltration was estimated using single-sample Gene Set Enrichment Analysis (ssGSEA). RESULTS: DNM1 expression in CC tissues was significantly higher than that in normal tissues. High DNM1 expression was significantly correlated with M stage, N stage, perineural invasion and lymphatic invasion and predicted poor prognosis. The univariate analysis highlighted that DNM1 was an independent CC risk factor. Results of ssGSEA showed that DNM1 was linked to several cancer-related pathways, including the neuroactive ligand-receptor interaction, hypertrophic cardiomyopathy, ECM-receptor interaction, dilated cardiomyopathy, and calcium signaling pathway. Moreover, DNM1 expression was positively correlated with the level of infiltration by Neutrophils, Tregs, NK cells, and Macrophages. CONCLUSION: DNM1 has a significant function and has diagnostic and prognostic potential for CC.

CryoEM structure of the super-constricted two-start dynamin 1 filament.

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network.

G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.

Altered Fast Synaptic Transmission in a Mouse Model of DNM1-Associated Developmental Epileptic Encephalopathy.

Developmental epileptic encephalopathies (DEEs) are severe seizure disorders that occur in infants and young children, characterized by developmental delay, cognitive decline, and early mortality. Recent efforts have identified a wide variety of genetic variants that cause DEEs. Among these, variants in the DNM1 gene have emerged as definitive causes of DEEs, including infantile spasms and Lennox-Gastaut syndrome. A mouse model of Dnm1-associated DEE, known as "Fitful" (Dnm1Ftfl ), recapitulates key features of the disease, including spontaneous seizures, early lethality, and neuronal degeneration. Previous work showed that DNM1 is a key regulator of synaptic vesicle (SV) endocytosis and synaptic transmission and suggested that inhibitory neurotransmission may be more reliant on DNM1 function than excitatory transmission. The Dnm1Ftfl variant is thought to encode a dominant negative DNM1 protein; however, the effects of the Dnm1Ftfl variant on synaptic transmission are largely unknown. To understand these synaptic effects, we recorded from pairs of cultured mouse cortical neurons and characterized all four major connection types [excitation of excitation (E-E), inhibition of inhibition (I-I), E-I, I-E]. Miniature and spontaneous EPSCs and IPSCs were larger, but less frequent, at all Dnm1Ftfl synaptic types, and Dnm1Ftfl neurons had reduced expression of excitatory and inhibitory SV markers. Baseline evoked transmission, however, was reduced only at inhibitory synapses onto excitatory neurons, because of a smaller pool of releasable SVs. In addition to these synaptic alterations, Dnm1Ftfl neurons degenerated later in development, although their activity levels were reduced, suggesting that Dnm1Ftfl may impair synaptic transmission and neuronal health through distinct mechanisms.

RNAi-Based Gene Therapy Rescues Developmental and Epileptic Encephalopathy in a Genetic Mouse Model.

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product.

Thyrotropin Causes Dose-dependent Biphasic Regulation of cAMP Production Mediated by Gs and Gi/o Proteins.

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.

Mutant p53 amplifies a dynamin-1/APPL1 endosome feedback loop that regulates recycling and migration.

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors downstream of gain-of-function (GOF) mutant p53. We report that dynamin-1 (Dyn1) is up-regulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Dyn1 is required for the recruitment and accumulation of the signaling scaffold, APPL1, to a spatially localized subpopulation of endosomes at the cell perimeter. We developed new tools to quantify peripherally localized early endosomes and measure the rapid recycling of integrins. We report that these perimeter APPL1 endosomes modulate Akt signaling and activate Dyn1 to create a positive feedback loop required for rapid recycling of EGFR and ß1 integrins, increased focal adhesion turnover, and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus that integrates signaling and receptor trafficking, which can be co-opted and amplified in mutant p53-driven cancer cells to increase migration and invasion.