Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.

Internalization of clathrin-coated vesicles from the plasma membrane constitutes the major endocytic route for receptors and their ligands. Dynamic and structural properties of endocytic clathrin coats are regulated by the mechanical properties of the plasma membrane. Here, we used conventional fluorescence imaging and multiple modes of structured illumination microscopy (SIM) to image formation of endocytic clathrin coats within live cells and tissues of developing fruit fly embryos. High resolution in both spatial and temporal domains allowed us to detect and characterize distinct classes of clathrin-coated structures. Aside from the clathrin pits and plaques detected in distinct embryonic tissues, we report, for the first time, formation of giant coated pits (GCPs) that can be up to two orders of magnitude larger than the canonical pits. In cultured cells, we show that GCP formation is induced by increased membrane tension. GCPs take longer to grow but their mechanism of curvature generation is the same as the canonical pits. We also demonstrate that GCPs split into smaller fragments during internalization. Considering the supporting roles played by actin filament dynamics under mechanically stringent conditions that slow down completion of clathrin coats, we suggest that local changes in the coat curvature driven by actin machinery can drive splitting and internalization of GCPs.

De novo endocytic clathrin coats develop curvature at early stages of their formation.

Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.

Dynamic interplay between cell membrane tension and clathrin-mediated endocytosis.

Deformability of the plasma membrane, the outermost surface of metazoan cells, allows cells to be dynamic, mobile and flexible. Factors that affect this deformability, such as tension on the membrane, can regulate a myriad of cellular functions, including membrane resealing, cell motility, polarisation, shape maintenance, membrane area control and endocytic vesicle trafficking. This review focuses on mechanoregulation of clathrin-mediated endocytosis (CME). We first delineate the origins of cell membrane tension and the factors that yield to its spatial and temporal fluctuations within cells. We then review the recent literature demonstrating that tension on the membrane is a fast-acting and reversible regulator of CME. Finally, we discuss tension-based regulation of endocytic clathrin coat formation during physiological processes.

Targeting Ras Activity Prevented Amyloid Beta-Induced Aberrant Neuronal Cell Cycle Re-Entry and Death.

Several studies suggest that soluble Amyloid ß (Aß) oligomer-induced aberrant neuronal cell cycle re-entry is the initial trigger for a significant part of the neuronal degeneration and loss in Alzheimer's disease (AD). In this study, we investigated the role of Ras, which is a well-known protooncoprotein, in soluble Aß oligomer-induced aberrant neuronal cell cycle activation and subsequent cell loss using retinoic acid differentiated human SH-SY5Y neuroblastoma cells as model system. In line with previous literature, we showed that in vitro preparations of soluble Aß42 oligomers triggered cell cycle activation but not cell proliferation. As a new finding, we showed that Farnesylthiosalicylic acid (FTS), a specific chemical Ras inhibitor, prevented soluble Aß42 oligomer preparation-induced cell cycle activation. Moreover, we showed that the expression of dominant negative mutant H-Ras (S17N) prevented soluble Aß42 oligomer preparation-induced cell cycle activation, confirming the specific role of Ras in this pathway. As a possible better mimic of the situation in the AD brain, we prepared soluble oligomers from Aß42 : Aß40 (3:7) peptide mixture and showed that this oligomer preparation similarly induced cell cycle activation which was also inhibited by the Ras inhibitor. Finally, we showed that FTS prevented soluble Aß42 oligomer preparationinduced cell death in our retinoic acid differentiated SH-SY5Y cells. Overall, our results strongly suggest that Ras activity is required for soluble Aß oligomer-induced aberrant neuronal cell cycle reentry and subsequent neuronal loss, which are considered important mechanisms in AD pathogenesis.

DDB1 and CUL4 associated factor 11 (DCAF11) mediates degradation of Stem-loop binding protein at the end of S phase.

In eukaryotes, bulk histone expression occurs in the S phase of the cell cycle. This highly conserved system is crucial for genomic stability and proper gene expression. In metazoans, Stem-loop binding protein (SLBP), which binds to 3' ends of canonical histone mRNAs, is a key factor in histone biosynthesis. SLBP is mainly expressed in S phase and this is a major mechanism to limit bulk histone production to the S phase. At the end of S phase, SLBP is rapidly degraded by proteasome, depending on two phosphorylations on Thr 60 and Thr 61. Previously, we showed that SLBP fragment (aa 51-108) fused to GST, is sufficient to mimic the late S phase (S/G2) degradation of SLBP. Here, using this fusion protein as bait, we performed pull-down experiments and found that DCAF11, which is a substrate receptor of CRL4 complexes, binds to the phosphorylated SLBP fragment. We further confirmed the interaction of full-length SLBP with DCAF11 and Cul4A by co-immunoprecipitation experiments. We also showed that DCAF11 cannot bind to the Thr61/Ala mutant SLBP, which is not degraded at the end of S phase. Using ectopic expression and siRNA experiments, we demonstrated that SLBP expression is inversely correlated with DCAF11 levels, consistent with the model that DCAF11 mediates SLBP degradation. Finally, we found that ectopic expression of the S/G2 stable mutant SLBP (Thr61/Ala) is significantly more toxic to the cells, in comparison to wild type SLBP. Overall, we concluded that CRL4-DCAF11 mediates the degradation of SLBP at the end of S phase and this degradation is essential for the viability of cells.

Translation regulation and proteasome mediated degradation cooperate to keep stem-loop binding protein low in G1-phase.

Histone mRNA levels are cell cycle regulated, and the major regulatory steps are at the posttranscriptional level. A major regulatory mechanism is S-phase restriction of Stem-loop binding protein (SLBP) which binds to the 3' end of histone mRNA and participates in multiple steps of histone mRNA metabolism, including 3' end processing, translation and regulation of mRNA stability. SLBP expression is cell cycle regulated without significant change in its mRNA level. SLBP expression is low in G1 until just before S phase where it functions and at the end of S phase SLBP is degraded by proteasome complex depending on phosphorylations on Thr60 and Thr61. Here using synchronized HeLa cells we showed that SLBP production rate is low in early G1 and recovers back to S phase level somewhere between early and mid-G1. Further, we showed that SLBP is unstable in G1 due to proteasome mediated degradation as a novel mechanism to keep SLBP low in G1. Finally, the S/G2 stable mutant form of SLBP is degraded by proteasome in G1, indicating that indicating that the SLBP degradation in G1 is independent of the previously identified SLBP degradation at S/G2. In conclusion, as a mechanism to limit histone production to S phase, SLBP is kept low in G1 phase due to cooperative action of translation regulation and proteasome mediated degradation which is independent of previously known S/G2 degradation.