Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.

The field of synthetic biology aims to design biological systems to perform tasks to better understand analogous natural systems and for direct applications in research and medicine (e.g., see Andrianantoandro et al. 2006;Drubin et al. 2007). Currently our ability to design biological systems is limited by the difficulty of predicting the behavior of even simple genetic circuits because often a given network topology will show qualitatively different behavior depending on the quantitative features of the underlying components. While advances have been made with networks composed of well-studied modules in bacteria (Guido et al. 2006), this challenge is acute in more complex eukaryotic systems where the components rarely have measured characteristics. Therefore, to make rapid progress in designing eukaryotic systems, synthetic biologists require both a pool of quantitatively annotated biological parts and the knowledge that these parts can be logically engineered into more complex networks with predictable function.One such network, which carries intrinsic value and tests a bottom-up design approach, is a network that confers memory. In this study we describe the rational design and construction of a high fidelity, modular memory device in yeast based on transcriptionally controlled autoregulatory positive feedback. This device heritably retains an induced state in individual cells in response to a transient stimulus. The rational design approach used here employs an extensive in vivo quantitative characterization of a set of synthetic transcription factors and the prediction of system behavior via network models incorporating these measured parameters. By successfully constructing this memory device, we established the essential parameters for maintaining an autoregulatory positive feedback loop in a dividing cellular system. Most importantly, we demonstrated predictability of system behavior in eukaryotes when the system is built from well-understood components. Results and Discussion Functional activators based on a modular architectureTo rationally engineer a memory device, we designed a set of fluorescently labeled synthetic transcription factors and their corresponding reporter genes to serve as candidate components. Each activator gene consists of a DNA-binding domain (DBD), two tandem copies of the monomeric red fluorescent protein (RFP) mCherry (Shaner et al. 2004), the viral activation domain VP64 (Beerli et al. 1998), and the SV40 nuclear localization sequence (NLS) (Kalderon et al. 1984;Lanford and Butel 1984), all under control of the GAL1/10 promoter (Fig. 1A). Each reporter gene has multiple copies of the DNAbinding sites corresponding to its given transcription factor upstream of the minimal CYC1 promoter, and its protein coding region encodes two tandem copies of the yellow fluorescent protein variant (YFP) Venus ( Fig. 1A; Nagai et al. 2002). The DBDs used were the LexA DBD (Hurstel et al. 1986(Hurstel et al. , 1988, an engineered variant of the murine zinc-finger Zif268 (ZifH) (Hurt et al. 200...

Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly.

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

A separation-of-function point mutant in APC is generated that disrupts actin nucleation without affecting APC’s other functions. Genetic analysis using this mutant reveals that APC stimulates actin assembly in living cells and that this activity is critical for microtubule-induced turnover of focal adhesions and directed cell migration.

Establishment and maintenance of silent chromatin in the Saccharomyces cerevisiae involves a step-wise assembly of the SIR complex. Here we demonstrate a role for the protein arginine methyltransferase Hmt1 in this process. In the absence of catalytically active Hmt1, yeast cells display increased transcription from silent chromatin regions and increased mitotic recombination within tandem repeats of rDNA. At the molecular level, loss of Hmt1's catalytic activity results in decreased Sir2 and dimethylated Arg-3 histone H4 occupancy across silent chromatin regions. These data suggest a model whereby protein arginine methylation affects the establishment and maintenance of silent chromatin. Received April 11, 2006; revised version accepted October 23, 2006. In a eukaryotic cell, different chromosomal regions display varying degrees of transcriptional competency. This is the result of selective transcriptional repression or silencing that renders specific chromatin domains inaccessible to the transcriptional machinery. Analogous to metazoan heterochromatin, there are three chromosomal regions in the yeast Saccharomyces cerevisiae that are epigenetically transcriptionally silenced (Rusche et al. 2003); the telomeres, the silent mating loci (HMR and HML), and the rDNA loci. Within these regions are cisacting elements known as silencers and telomeric repeats that serve as nucleation points to recruit transacting proteins responsible for the establishment and maintenance of silent chromatin.One of the most prominent trans-acting proteins involved in the establishment and maintenance of silencing at these loci is Silent Information Regulator-2 (Sir2), an NAD + -dependent histone deacetylase (Blander and Guarente 2004). Sir2 plays a crucial role in the formation of silent chromatin. In addition, Sir2 is involved in the maintenance of genome stability via the repair of doublestranded DNA breaks by nonhomologous recombination. For example, Sir2 is recruited to sites of DNA strand breaks (Martin et al. 1999;Mills et al. 1999) and Sir2-dependent localized histone acetylation triggers the homologous recombination pathway of double-strand DNA repair (Tamburini and Tyler 2005).Sir2 forms complexes with different proteins to promote silencing. For example, the Sir1/2/3/4 complex functions to silence transcription at the mating-type loci, whereas a Sir2/3/4 complex mediates silencing at the telomeres (Kaeberlein et al. 1999). At the rDNA, Sir2 associates with Cdc14 and Net1 to form the RENT (regulator of the nucleolar silencing and telophase exit) complex (Ghidelli et al. 2001). These complexes are thought to promote the formation of silent chromatin through stepwise propagation along the DNA (Hoppe et al. 2002).Arginine methylation is a post-translational modification commonly found in nucleic acid-binding proteins (Bedford and Richard 2005). The enzyme that catalyzes this modification belongs to a growing enzyme family called protein arginine methyltransferases or PRMTs. Arginine residues may be either monomethylated or di...

A new in vivo role is defined for the yeast F-BAR protein Hof1 in polarized cell growth. Hof1 dimers bind to the FH1 domains of the formin Bnr1 and inhibit actin polymerization while the formin remains attached to filament ends. This activity is required in vivo for normal actin cable architecture and polarized secretion.

Combined use of genetics and quantitative cell imaging reveals that Smy1 and Bud14 use common sequence motifs to directly regulate formins in vivo and thereby assemble actin cable structures of a particular shape and velocity to support efficient transport of secretory vesicles.