Cell surface receptor uptake via clathrin-mediated endocytosis (CME) and subsequent intracellular sorting for degradation or recycling regulates the strength and specificity of downstream signaling. ...Signaling, in turn, modulates early endocytic trafficking. This reciprocal regulation of signaling and endocytosis provides opportunities for the establishment of feedback loops to enhance or suppress surface-derived signals. Recent studies suggest that dynamin-1, a presumed neuron-specific isoform of the large, membrane fission GTPase, can be activated in nonneuronal cells downstream of cancer-relevant signaling pathways and thereby function as a nexus between signaling and early endocytic trafficking. I speculate that sustained up-regulation and/or acute activation of dynamin-1 in cancer cells contributes to a program of "adaptive" CME that alters signaling to enhance cancer cell survival, migration, and proliferation.
Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 ...(AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.
Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo ...uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.
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•Highly sensitive detection of protein association with CCPs•CCPs undergo a multistep maturation process gated by a checkpoint•Early dynamin recruitment is required for CCP maturation•Compensatory mechanisms obscure EAP roles in CCP stabilization and maturation
Aguet et al. perform a high-sensitivity analysis of clathrin-coated pits (CCPs). They establish methods that circumvent many difficulties associated with low signal-to-noise ratios, enable quantitative tracking of CCP/accessory dynamics, and reveal that dynamin and the α-adaptin appendage domain of AP2 each gate different stages in the progression of clathrin-mediated endocytosis.
Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane ...composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.
The dynamin superfamily Ramachandran, Rajesh; Schmid, Sandra L.
Current biology,
04/2018, Letnik:
28, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The dynamin superfamily comprises a growing assortment of multi-domain GTPases, found from bacteria to man, that are distinguished from typical GTPases of the Ras, Rab and G-protein families by their ...modular structure (Figure 1), relatively large size (>70 kDa), and low affinity for guanine nucleotides. In addition, they display a conserved propensity to self-assemble into polymeric arrays, the dynamics of which are regulated by an autonomous, assembly-stimulated GTPase activity.
Here, Ramachandran and Schmid describe the structural, biophysical, and biochemical properties of the dynamin superfamily and the implications for their distinct cellular functions.
Sorting nexin (SNX)9 was first discovered as an endocytic accessory protein involved in clathrin-mediated endocytosis. However, recent data suggest that SNX9 is a multifunctional scaffold that ...coordinates membrane trafficking and remodeling with changes in actin dynamics to affect diverse cellular processes. Here, we review the accumulated knowledge on SNX9 with an emphasis on its recently identified roles in clathrin-independent endocytic pathways, cell invasion, and cell division, which have implications for SNX9 function in human disease, including cancer.
The San Francisco Declaration on Research Assessment (DORA) was penned 5 years ago to articulate best practices for how we communicate and judge our scientific contributions. In particular, it ...adamantly declared that Journal Impact Factor (JIF) should never be used as a surrogate measure of the quality of individual research contributions, or for hiring, promotion, or funding decisions
Since then, a heightened awareness of the damaging practice of using JIFs as a proxy for the quality of individual papers, and to assess an individual's or institution's accomplishments has led to changes in policy and the design and application of best practices to more accurately assess the quality and impact of our research. Herein I summarize the considerable progress made and remaining challenges that must be met to ensure a fair and meritocratic approach to research assessment and the advancement of research.
The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process ...remain a mystery. We present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2 Å and a fusion protein, GG, linking human dynamin 1's catalytic G domain to its GTPase effector domain (GED) at 2.2 Å. The structures reveal the position and connectivity of dynamin fragments in the assembled structure, showing that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are made of two dimers, in which the G domain of one molecule interacts in
trans with the GED of another. Structural comparison of GG
GMPPCP to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane-remodeling events necessary for fission.
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► CryoEM structure of dynamin polymer shows subunit interactions ► Tetramer is two dimers, in which GED domain of one dynamin binds G domain of another ► Structure of G domain–GED fusion shows hydrolysis-dependent conformational changes ► The conformational changes create a powerstroke that may drive membrane fission
The GTPase dynamin forms collars around vesicle necks and catalyzes membrane fission. Structural studies reveal that dynamin dimerization and GTP hydrolysis trigger large conformational changes that may drive membrane fission.
Dynamin, best studied for its role in clathrin-mediated endocytosis, is the prototypical member of a family of multidomain GTPases involved in fission and remodeling of multiple organelles. Recent ...studies have shown that dynamin alone can catalyze fission of membrane tubules and vesicle formation from planar lipid templates. Thus, dynamin appears to be a self-sufficient fission machine. Here we review the biochemical activities and structural features of dynamin required for fission activity. As all changes in membrane topology require energetically unfavorable rearrangements of the lipid bilayer, we discuss the interplay between dynamin and its lipid substrates that are critical to defining a nonleaky pathway to membrane fission. We propose a two-stage model for dynamin-catalyzed fission. In stage one, dynamin's mechanochemical activities induce localized curvature stress and position its lipid-interacting pleckstrin homology domains to create a catalytic center that, in stage two, guides lipid remodeling through hemifission intermediates to drive membrane fission.
The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated ...pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.
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