scholarly journals Tubular microdomains of Rab7-endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth 2B

2021 ◽  
Author(s):  
R. Markworth ◽  
V. Dambeck ◽  
L.M. Steinbeck ◽  
A. Koufali ◽  
B. Bues ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Neuronal Cultures ◽  
Charcot Marie Tooth ◽  
Late Endosomes ◽  
Receptor Kinases ◽  
Retrieval Mechanism ◽  
Survival And Growth ◽  
Challenging Question ◽  
Neurotrophic Receptors ◽  
Insulating Properties

Axonal survival and growth requires signalling from tropomyosin receptor kinases (Trks). To transmit their signals, receptor-ligand complexes are endocytosed and retrogradely trafficked to the soma where downstream signalling occurs. Vesicles transporting neurotrophic receptors to the soma are reported to be Rab7-positive late endosomes/multi vesicular bodies where receptors localize within so-called intraluminal vesicles. Therefore, one challenging question is how downstream signalling is possible given the insulating properties of intraluminal vesicles. In this study, we report that Rab7-endosomes/multi vesicular bodies retrieve TrkA through tubular microdomains. Interestingly, this phenotype is absent for the EGF-receptor. Further, we found that EndophilinA1, EndophilinA2 and EndophilinA3 together with WASH1 are involved in the tubulation process. In Charcot-Marie-Tooth 2B, a neuropathy of the peripheral nervous system, this tubulating mechanism is disrupted. In addition, the ability to tubulate correlates with the phosphorylation levels of TrkA as well as with neurite length in neuronal cultures from dorsal root ganglia. In all, we report a new retrieval mechanism of late Rab7-endosomes, which enables TrkA signalling and sheds new light onto how neurotrophic signalling is disrupted in CMT2B.

scholarly journals Dysregulated Inflammatory Signaling upon Charcot-Marie-Tooth Type 1C Mutation of SIMPLE Protein

2015 ◽  
Vol 35 (14) ◽  
pp. 2464-2478 ◽  
Author(s):  
Wenjing Li ◽  
Hong Zhu ◽  
Xuelian Zhao ◽  
Deborah Brancho ◽  
Yuanxin Liang ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Signal Propagation ◽  
Transforming Growth Factor Β ◽  
Receptor Activation ◽  
Nuclear Accumulation ◽  
Endosomal Trafficking ◽  
Inflammatory Signaling ◽  
Charcot Marie Tooth ◽  
Late Endosomes ◽  
Tooth Type

Endosomal trafficking is a key mechanism to modulate signal propagation and cross talk. Ubiquitin adaptors, along withendosomalsortingcomplexrequired fortransport (ESCRT) complexes, are also integrated to terminate ligand-receptor activation in late endosomes and multivesicular bodies (MVBs). Within these pathways, we recently demonstrated that the protein SIMPLE is a novel player in MVB regulation. SIMPLE is also clinically important and its mutation accounts for the Charcot-Marie-Tooth type 1C (CMT1C) disease. MVB defects of mutation and deletion of SIMPLE, however, are distinct. Here, we show that MVB defects found in mutation but not deletion of SIMPLE lead to impaired turnover and accumulation of ESCRT-0 protein Hrs puncta in late endosomes. We further uncover increased colocalization of ubiquitin ligase TRAF6 and Hrs in late endosomes. Upon stimulation with interkeukin-1 or transforming growth factor β, prolonged activation of p38 kinase/JNK is detected, while nuclear accumulation of NF-κB and phosphorylation of SMAD2 is reduced with CMT1C mutation. The aberrant kinetics we observed in inflammatory signaling may contribute to increased tumor susceptibility and changes in the levels of chemokines/cytokines that result from CMT1C mutation. We propose that altered endosomal trafficking due to malformations of MVBs and subsequent atypical signaling kinetic may account for a toxic gain of function in CMT1C pathogenesis.

scholarly journals Role of Protein Kinase C βII in Influenza Virus Entry via Late Endosomes

2003 ◽  
Vol 77 (1) ◽  
pp. 460-469 ◽  
Author(s):  
Sara B. Sieczkarski ◽  
H. Alex Brown ◽  
Gary R. Whittaker
Keyword(s):  
Influenza Virus ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Egf Receptor ◽  
Virus Entry ◽  
Influenza Virus Infection ◽  
Target Cells ◽  
Mutant Form ◽  
Kinase C ◽  
Late Endosomes

ABSTRACT Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C βII (PKCβII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCα and -β. Cells expressing a phosphorylation-deficient form of PKCβII (T500V), but not an equivalent mutant form of PKCα, inhibited successful influenza virus entry—with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCβII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCβII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCβII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCβII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.

scholarly journals Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy

2018 ◽  
Vol 4 (3) ◽  
pp. e244 ◽  
Author(s):  
Markus T. Sainio ◽  
Emil Ylikallio ◽  
Laura Mäenpää ◽  
Jenni Lahtela ◽  
Pirkko Mattila ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Early Onset ◽  
Messenger Rna ◽  
Patient Specific ◽  
Neuronal Cultures ◽  
Cultured Neurons ◽  
Loss Of Function ◽  
Nonsense Mutations ◽  
Neurofilament Light ◽  
Charcot Marie Tooth

ObjectiveWe used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease.MethodsMotor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons.ResultsWe show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA.ConclusionsThe strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.

scholarly journals Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways.

1991 ◽  
Vol 112 (1) ◽  
pp. 55-63 ◽  
Author(s):  
A Sorkin ◽  
S Krolenko ◽  
N Kudrjavtceva ◽  
J Lazebnik ◽  
L Teslenko ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Relative Rate ◽  
Growth Factor Receptor ◽  
Temperature Sensitive ◽  
Egf Receptors ◽  
A431 Cells ◽  
Receptor Complexes ◽  
Late Endosomes ◽  
Long Time ◽  
Epidermal Growth

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.

scholarly journals In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

2008 ◽  
Vol 19 (11) ◽  
pp. 4942-4955 ◽  
Author(s):  
Thomas Falguières ◽  
Pierre-Philippe Luyet ◽  
Christin Bissig ◽  
Cameron C. Scott ◽  
Marie-Claire Velluz ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Light And Electron Microscopy ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Vesicle Budding ◽  
Late Endosomes ◽  
Electron Microscopy Analysis ◽  
Membrane Components

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.

scholarly journals A dysfunctional endolysosomal pathway common to two sub-types of demyelinating Charcot–Marie–Tooth disease

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
James R. Edgar ◽  
Anita K. Ho ◽  
Matilde Laurá ◽  
Rita Horvath ◽  
Mary M. Reilly ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Loss Of Function ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
Recessive Mutations ◽  
Late Endosomes ◽  
Demyelinating Peripheral Neuropathy ◽  
Tooth Disease

Abstract Autosomal dominant mutations in LITAF are responsible for the rare demyelinating peripheral neuropathy, Charcot–Marie–Tooth disease type 1C (CMT1C). The LITAF protein is expressed in many human cell types and we have investigated the consequences of two different LITAF mutations in primary fibroblasts from CMT1C patients using confocal and electron microscopy. We observed the appearance of vacuolation/enlargement of late endocytic compartments (late endosomes and lysosomes). This vacuolation was also observed after knocking out LITAF from either control human fibroblasts or from the CMT1C patient-derived cells, consistent with it being the result of loss-of-function mutations in the CMT1C fibroblasts. The vacuolation was similar to that previously observed in fibroblasts from CMT4J patients, which have autosomal recessive mutations in FIG4. The FIG4 protein is a component of a phosphoinositide kinase complex that synthesises phosphatidylinositol 3,5-bisphosphate on the limiting membrane of late endosomes. Phosphatidylinositol 3,5-bisphosphate activates the release of lysosomal Ca2+ through the cation channel TRPML1, which is required to maintain the homeostasis of endosomes and lysosomes in mammalian cells. We observed that a small molecule activator of TRPML1, ML-SA1, was able to rescue the vacuolation phenotype of LITAF knockout, FIG4 knockout and CMT1C patient fibroblasts. Our data describe the first cellular phenotype common to two different subtypes of demyelinating CMT and are consistent with LITAF and FIG4 functioning on a common endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes. Although our experiments were on human fibroblasts, they have implications for our understanding of the molecular pathogenesis and approaches to therapy in two subtypes of demyelinating Charcot–Marie–Tooth disease.

scholarly journals Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases

2005 ◽  
Vol 102 (31) ◽  
pp. 11011-11016 ◽  
Author(s):  
T. A. Carter ◽  
L. M. Wodicka ◽  
N. P. Shah ◽  
A. M. Velasco ◽  
M. A. Fabian ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Drug Resistant ◽  
Receptor Kinases ◽  
Resistant Mutants

scholarly journals The ESCRT-III Subunit hVps24 Is Required for Degradation but Not Silencing of the Epidermal Growth Factor Receptor

2006 ◽  
Vol 17 (6) ◽  
pp. 2513-2523 ◽  
Author(s):  
Kristi G. Bache ◽  
Susanne Stuffers ◽  
Lene Malerød ◽  
Thomas Slagsvold ◽  
Camilla Raiborg ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Susceptibility Gene ◽  
Growth Factor Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Endosomal Sorting ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Epidermal Growth

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

scholarly journals The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes

2011 ◽  
Vol 22 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Dafne Chirivino ◽  
Laurence Del Maestro ◽  
Etienne Formstecher ◽  
Philippe Hupé ◽  
Graça Raposo ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Protein Sorting ◽  
Small Gtpases ◽  
Erm Proteins ◽  
Degradative Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
In Cells ◽  
Hops Complex

In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.

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