The evolution of dynamin to regulate clathrin-mediated endocytosis: speculations on the evolutionarily late appearance of dynamin relative to clathrin-mediated endocytosis

Whereas clathrin-mediated endocytosis (CME) exists in all eukaryotic cells, we first detect classical dynamin in Ichthyosporid, a single-cell, metazoan precursor. Based on a key functional residue in its pleckstrin homology domain, we speculate that the evolution of metazoan dynamin coincided with the specialized need for regulated CME during neurotransmission.     

Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin‐mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin‐coated pits and bulk endocytosis‐like structures increase on the plasma membrane in Syt11‐knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.     

Tau overexpression impairs neuronal endocytosis by decreasing the GTPase dynamin 1 through the miR‐132/MeCP2 pathway

Tauopathies are a class of neurodegenerative diseases that are characterized by pathological aggregation of tau protein, which is accompanied by synaptic disorders. However, the role of tau in endocytosis, a fundamental process in synaptic transmission, remains elusive. Here, we report that forced expression of human tau (hTau) in mouse cortical neurons impairs endocytosis by decreasing the level of the GTPase dynamin 1 via disruption of the miR‐132‐MeCP2 pathway; this process can also be detected in the brains of Alzheimer's patients and hTau mice. Our results provide evidence for a novel role of tau in the regulation of presynaptic function.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

Regulation of dynamic polarity switching in bacteria by a Ras‐like G‐protein and its cognate GAP

The rod‐shaped cells of the bacterium Myxococcus xanthus move uni‐directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. Cellular reversals depend on pole‐to‐pole relocation of motility proteins that localize to the cell poles between reversals. We show that MglA is a Ras‐like G‐protein and acts as a nucleotide‐dependent molecular switch to regulate motility and that MglB represents a novel GTPase‐activating protein (GAP) family and is the cognate GAP of MglA. Between reversals, MglA/GTP is restricted to the leading and MglB to the lagging pole defining the leading/lagging polarity axis. For reversals, the Frz chemosensory system induces the relocation of MglA/GTP to the lagging pole causing an inversion of the leading/lagging polarity axis. MglA/GTP stimulates motility by establishing correct polarity of motility proteins between reversals and reversals by inducing their pole‐to‐pole relocation. Thus, the function of Ras‐like G‐proteins and their GAPs in regulating cell polarity is found not only in eukaryotes, but also conserved in bacteria.     

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1‐like protein

Dynamin 1‐like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.     

Compensatory endocytosis in bladder umbrella cells occurs through an integrin‐regulated and RhoA‐ and dynamin‐dependent pathway

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin‐independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding‐stimulated CE, which depended on β₁ integrin‐associated signalling pathways, occurred by a dynamin‐, actin‐, and RhoA‐regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO‐1‐positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin‐independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA‐ and dynamin‐dependent pathway, and terminates in degradation and not recapture of membrane in DFV.     

Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481-486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.     

Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics

The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.     

ARAP1 regulates endocytosis of EGFR

Signaling through the EGF receptor is regulated by endocytosis. ARAP1 is a protein with Arf guanosine triphosphatase-activating protein (GAP) and Rho GAP domains. We investigated the role of ARAP1 in EGF receptor endocytic trafficking. Following EGF treatment of cells, ARAP1 rapidly and transiently associated with the edge of the cell and punctate structures containing Rab5, rabaptin 5 and EGFR but not early embryonic antigen 1 (EEA1). EGF associated with the ARAP1-positive punctate structures prior to EEA1-positive early endosomes. Recruitment of ARAP1 to the punctate structures required active Rab5 and an additional signal from EGFR. Decreasing ARAP1 levels with small interfering RNA accelerated association of EGF with EEA1 endosomes and degradation of EGFR. Phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun-amino-terminal kinase (JNK) was diminished and more transient in cells with reduced levels of ARAP1 than in controls. Based on these findings, we propose that ARAP1 regulates the endocytic traffic of EGFR and, consequently, the rate of EGFR signal attenuation.     

Ikarugamycin: A Natural Product Inhibitor of Clathrin‐Mediated Endocytosis

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low‐density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin‐mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC₅₀ of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long‐term incubation with IKA has cytotoxic effects, the short‐term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.      

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles

Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Because maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions, single SV retrieval modes such as clathrin-mediated endocytosis predominate. However, during increased neuronal activity, additional SV retrieval capacity is required, which is provided by activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mechanism during elevated neuronal activity. It is a high capacity SV retrieval mode that is immediately triggered during such stimulation conditions. This review will summarize the current knowledge regarding the molecular mechanism of ADBE, including molecules required for its triggering and subsequent steps, including SV budding from bulk endosomes. The molecular relationship between ADBE and the SV reserve pool will also be discussed. It is becoming clear that an understanding of the molecular physiology of ADBE will be of critical importance in attempts to modulate both normal and abnormal synaptic function during intense neuronal activity.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Structural insights of Mycobacterium GTPase‐Obg and anti‐sigma‐F factor Usfx interaction

An essential protein for bacterial growth, GTPase‐Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti‐sigma‐F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above‐mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg‐Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg‐Usfx interaction. Surface plasmon resonance results clearly suggest that N‐terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.     

Tctex‐1 controls ciliary resorption by regulating branched actin polymerization and endocytosis

The primary cilium is a plasma membrane‐protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin‐rich, endocytosis‐active periciliary subdomain. Furthermore, Tctex‐1, originally identified as a cytoplasmic dynein light chain, has a dynein‐independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex‐1, actin, and dynamin. Mechanistically, Tctex‐1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex‐1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin‐dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex‐1‐regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.     

Dynamin self-assembly and the vesicle scission mechanism: how dynamin oligomers cleave the membrane neck of clathrin-coated pits during endocytosis

Recently, Gao et al. and Chappie et al. elucidated the crystal structures of the polytetrameric stalk domain of the dynamin-like virus resistance protein, MxA, and of the G-domain dimer of the large, membrane-deforming GTPase, dynamin, respectively. Combined, they provide a hypothetical oligomeric structure for the complete dynamin protein. Here, it is discussed how the oligomers are expected to form and how they participate in dynamin mediated vesicle fission during the process of endocytosis. The proposed oligomeric structure is compared with the novel mechanochemical model of dynamin function recently proposed by Bashkirov et al. and Pucadyil and Schmid. In conclusion, the new model of the dynamin oligomer has the potential to explain how short self-limiting fissogenic dynamin assemblies are formed and how concerted GTP hydrolysis is achieved. The oligomerisation of two other dynamin superfamily proteins, the guanylate binding proteins (GBPs) and the immunity-related GTPases (IRGs), is addressed briefly.     

900 MHz modulated electromagnetic fields accelerate the clathrin‐mediated endocytosis pathway

We report new data regarding the molecular mechanisms of GSM‐induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 µs pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM–EMF/electric pulses in the presence of drugs inhibiting the clathrin‐ or the caveolin‐dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5–29.5 °C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin‐dependent endocytosis is stimulated by the GSM–EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin‐coated vesicles. Bioelectromagnetics 30:222–230, 2009. © 2008 Wiley‐Liss, Inc.     

Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.