A transmembrane serine residue in the Rot1 protein is essential for yeast cell viability.

Polar residues are present in TM (transmembrane) helices and may influence the folding or association of membrane proteins. In the present study, we use an in vivo approach to analyse the functional and structural roles for amino acids in membrane-spanning motifs using the Rot1 (reversal of Tor2 lethality 1) protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and it contains a single TM domain. An alanine insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical serine residue inside the helix that plays an essential role in maintaining cell viability in S. cerevisiae. Replacement of the serine residue at position 250 with a broad variety of amino acids did not affect protein targeting and location, but completely disrupted protein function causing cell death. Interestingly, substitution of the serine residue by threonine resulted in sustained cell viability, demonstrating that the hydroxy group of the TM serine side chain plays a critical role in protein function. The results of the present study indicate that Rot1 needs the TM Ser250 to interact with other membrane components and exert its functional role, avoiding exposure of the serine hydrogen-bonding group at the lipid-exposed surface.

Tumor invasiveness is regulated by the concerted function of APC, formins, and Arp2/3 complex.

Tumor cell invasion is the initial step in metastasis, the leading cause of death from cancer. Invasion requires protrusive cellular structures that steer the migration of leader cells emanating from the tumor mass toward neighboring tissues. Actin is central to these processes and is therefore the prime target of drugs known as migrastatics. However, the broad effects of general actin inhibitors limit their therapeutic use. Here, we delineate the roles of specific actin nucleators in tuning actin-rich invasive protrusions and pinpoint potential pharmacological targets. We subject colorectal cancer spheroids embedded in collagen matrix-a preclinical model mirroring solid tumor invasiveness-to pharmacologic and/or genetic treatment of specific actin arrays to assess their roles in invasiveness. Our data reveal coordinated yet distinct involvement of actin networks nucleated by adenomatous polyposis coli, formins, and actin-related protein 2/3 complex in the biogenesis and maintenance of invasive protrusions. These findings may open avenues for better targeted therapies.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

The compass to follow: Focal adhesion turnover.

How cells move is a fundamental biological question. The directionality of adherent migrating cells depends on the assembly and disassembly (turnover) of focal adhesions (FAs). FAs are micron-sized actin-based structures that link cells to the extracellular matrix. Traditionally, microtubules have been considered key to triggering FA turnover. Through the years, advancements in biochemistry, biophysics, and bioimaging tools have been invaluable for many research groups to unravel a variety of mechanisms and molecular players that contribute to FA turnover, beyond microtubules. Here, we discuss recent discoveries of key molecular players that affect the dynamics and organization of the actin cytoskeleton to enable timely FA turnover and consequently proper directed cell migration.

Confocal Laser Scanning Imaging of Cell Junctions in Human Colon Cancer Cells.

The intestinal epithelium is formed by a single layer of cells. These cells originate from self-renewal stem cells that give rise to various lineages of cells: Paneth, transit-amplifying, and fully differentiated cells (as enteroendocrine, goblet cells, and enterocytes). Enterocytes, also known as absorptive epithelial cells, are the most abundant cell type in the gut. Enterocytes have the potential to polarize as well as form tight junctions with neighbor cells which altogether serve to ensure both the absorption of "good" substances into the body and the blockage of "bad" substances, among other functions. Culture cell models such as the Caco-2 cell line have been proved to be valuable tools to study the fascinating functions of the intestine. In this chapter we outline some experimental procedures to grow, differentiate, and stain intestinal Caco-2 cells, as well as image them using two modes of confocal laser scanning microscopy.

Automated Quantitative Analysis of Shape Features in Human Epithelial Monolayers and Spheroids Generated from Colorectal Cancer Cells.

Advancements in microscopy techniques permit us to acquire endless datasets of images. A major bottleneck in cell imaging is how to analyze petabytes of data in an effective, reliable, objective, and effortless way. Quantitative imaging is becoming crucial to disentangle the complexity of many biological and pathological processes. For instance, cell shape is a summary readout of a myriad of cellular processes. Changes in cell shape use to reflect changes in growth, migration mode (including speed and persistence), differentiation stage, apoptosis, or gene expression, serving to predict health or disease. However, in certain contexts, e.g., tissues or tumors, cells are tightly packed together, and measurement of individual cellular shapes can be challenging and laborious. Bioinformatics solutions like automated computational image methods provide a blind and efficient analysis of large image datasets. Here we describe a detailed and friendly step-by-step protocol to extract various cellular shape parameters quickly and accurately from colorectal cancer cells forming either monolayers or spheroids. We envision those similar settings could be extended to other cell lines, colorectal and beyond, either label/unlabeled or in 2D/3D environments.

APC-driven actin nucleation powers collective cell dynamics in colorectal cancer cells.

Cell remodeling relies on dynamic rearrangements of cell contacts powered by the actin cytoskeleton. The tumor suppressor adenomatous polyposis coli (APC) nucleate actin filaments (F-actin) and localizes at cell junctions. Whether APC-driven actin nucleation acts in cell junction remodeling remains unknown. By combining bioimaging and genetic tools with artificial intelligence algorithms applied to colorectal cancer cell, we found that the APC-dependent actin pool contributes to sustaining levels of F-actin, as well as E-cadherin and occludin protein levels at cell junctions. Moreover, this activity preserved cell junction length and angle, as well as vertex motion and integrity. Loss of this F-actin pool led to larger cells with slow and random cell movement within a sheet. Our findings suggest that APC-driven actin nucleation promotes cell junction integrity and dynamics to facilitate collective cell remodeling and motility. This offers a new perspective to explore the relevance of APC-driven cytoskeletal function in gut morphogenesis.

Cytoskeletal Control and Wnt Signaling-APC's Dual Contributions in Stem Cell Division and Colorectal Cancer.

Intestinal epithelium architecture is sustained by stem cell division. In principle, stem cells can divide symmetrically to generate two identical copies of themselves or asymmetrically to sustain tissue renewal in a balanced manner. The choice between the two helps preserve stem cell and progeny pools and is crucial for tissue homeostasis. Control of spindle orientation is a prime contributor to the specification of symmetric versus asymmetric cell division. Competition for space within the niche may be another factor limiting the stem cell pool. An integrative view of the multiple links between intracellular and extracellular signals and molecular determinants at play remains a challenge. One outstanding question is the precise molecular roles of the tumour suppressor Adenomatous polyposis coli (APC) for sustaining gut homeostasis through its respective functions as a cytoskeletal hub and a down regulator in Wnt signalling. Here, we review our current understanding of APC inherent activities and partners in order to explore novel avenues by which APC may act as a gatekeeper in colorectal cancer and as a therapeutic target.

The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.

Methods of Synchronization of Yeast Cells for the Analysis of Cell Cycle Progression.

Cell division is a fascinating and fundamental process that sustains life. By this process, unicellular organisms reproduce and multicellular organisms sustain development, growth, and tissue repair. Division of a mother cell gives rise to two daughter cells according to an ordered set of events within four successive phases called G1 (gap1), S (DNA Synthesis), G2 (gap2), and M (Mitosis) phase. How these different phases are orchestrated to ensure the physical separation of the two daughter cells is a tightly regulated process. Indeed, inappropriate cell division could lead to uncontrolled cell proliferation and ultimately to cancer. Saccharomyces cerevisiae is an excellent model system for unraveling the secrets of cell division. A large community of researchers has chosen budding yeast as a model because of its advantages: rapid growth in simple and economical media, tractable genetics, powerful biochemistry, cell biology, and proteomics approaches. Furthermore, the cell cycle mechanisms, as elucidated in yeast, are conserved in higher eukaryotes. The ability to synchronize and get large numbers of cells in a particular stage of the cell cycle is crucial to properly explore the mechanisms of the cell cycle. An overview of the most common yeast synchronization techniques has been compiled in this chapter.

Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis.

Profilin is an abundant actin monomer-binding protein with critical actin regulatory roles in vivo [1, 2]. However, profilin also influences microtubule dynamics in cells, which may be mediated in part through its interactions with formins that in turn bind microtubules [3, 4]. Specific residues on human profilin-1 (PFN1) are mutated in patients with amyotrophic lateral sclerosis (ALS) [5, 6]. However, the observation that some ALS-linked PFN1 mutants fail to alter cellular actin organization or dynamics [5-8] or in vitro actin-monomer affinity [9] has been perplexing, given that profilin is best understood as an actin regulator. Here, we investigated direct effects of profilin on microtubule dynamics and whether ALS-linked mutations in PFN1 disrupt such functions. We found that human, fly, and yeast profilin homologs all directly enhance microtubule growth rate by several-fold in vitro. Microtubule stimulatory effects were unaffected by mutations in the canonical actin- or poly-proline-binding sites of profilin. Instead, microtubule activities depended on specific surface residues on profilin mutated in ALS patients. Furthermore, microtubule effects were attenuated by increasing concentrations of actin monomers, suggesting competition between actin and microtubules for binding profilin. Consistent with these biochemical observations, a 2-fold increase in the expression level of wild-type PFN1, but not the ALS-linked PFN1 mutants, increased microtubule growth rates in cells. Together, these results demonstrate that profilin directly enhances the growth rate of microtubules. They further suggest that ALS-linked mutations in PFN1 may perturb cellular microtubule dynamics and/or the coordination between the actin and microtubule cytoskeletons, leading to motor neuron degeneration.

Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover.

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT-actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration.

Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2.

Formins are widespread actin-polymerizing proteins that play pivotal roles in a number of processes, such as cell polarity, morphogenesis, cytokinesis, and cell migration. In agreement with their crucial function, formins are prone to a variety of regulatory mechanisms that include autoinhibition, post-translational modifications, and interaction with formin modulators. Furthermore, activation and function of formins is intimately linked to their ability to interact with membranes. In the budding yeast Saccharomyces cerevisiae, the two formins Bni1 and Bnr1 play both separate and overlapping functions in the organization of the actin cytoskeleton. In addition, they are controlled by both common and different regulatory mechanisms. Here we show that proper localization of both formins requires the redundant E3 ubiquitin ligases Dma1 and Dma2, which were previously involved in spindle positioning and septin organization. In dma1 dma2 double mutants, formin distribution at polarity sites is impaired, thus causing defects in the organization of the actin cable network and hypersensitivity to the actin depolymerizer latrunculin B. Expression of a hyperactive variant of Bni1 (Bni1-V360D) rescues these defects and partially restores proper spindle positioning in the mutant, suggesting that the failure of dma1 dma2 mutant cells to position the spindle is partly due to faulty formin activity. Strikingly, Dma1/2 interact physically with both formins, while their ubiquitin-ligase activity is required for formin function and polarized localization. Thus, ubiquitylation of formin or a formin interactor(s) could promote formin binding to membrane and its ability to nucleate actin. Altogether, our data highlight a novel level of formin regulation that further expands our knowledge of the complex and multilayered controls of these key cytoskeleton organizers.

Spindle pole body history intrinsically links pole identity with asymmetric fate in budding yeast.

BACKGROUND: Budding yeast is a unique model for exploring differential fate in a cell dividing asymmetrically. In yeast, spindle orientation begins with the old spindle pole body (SPB) (from the preceding cell cycle) contacting the bud by its existing astral microtubules (aMTs) while the new pole delays astral microtubule organization. This appears to prime the inheritance of the old pole by the bud. The basis for this asymmetry and the discrimination of the poles by virtue of their history remain a mystery. RESULTS: Here, we report that asymmetric aMT organization stems from an outstanding structural asymmetry linked to the SPB cycle. We show that the γ-tubulin nucleation complex (γTC) favors the old spindle pole, an asymmetry inherent to the outer plaque (the cytoplasmic face of the SPB). Indeed, Spc72 (the receptor for the γTC) is acquired by the new SPB outer plaque partway through spindle assembly. The significance of this asymmetry was explored in cells expressing an Spc72(1-276)-Cnm67 fusion that forced symmetric nucleation at the SPB outer plaques. This manipulation triggered simultaneous aMT organization by both spindle poles from the outset and led to symmetric contacts between poles and the bud, effectively disrupting the program for spindle polarity. Temporally symmetric aMT organization perturbed Kar9 polarization by randomizing the choice of the pole to be guided toward the bud. Accordingly, the pattern of SPB inheritance was also randomized. CONCLUSIONS: Spc72 differential recruitment imparting asymmetric aMT organization represents the most upstream determinant linking SPB historical identity and fate.

Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae.

BACKGROUND: Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G(1). Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. RESULTS: We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. CONCLUSIONS: Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors-Kip3p and dynein.

Ase1p phosphorylation by cyclin-dependent kinase promotes correct spindle assembly in S. cerevisiae.

Spindle morphogenesis and dynamics follow an orderly sequence of events coupled to the oscillatory activation of cyclin-dependent kinase (CDK). Using S. cerevisiae, we have addressed the requirement of CDK for phosphorylation of the spindle midzone component Ase1p and its significance to spindle assembly. Ase1p is related to human PRC1, a protein negatively regulated by CDK until late mitosis, when it is required for central spindle organization and cytokinesis. By contrast, we show here that Ase1p phosphorylation by CDK promotes correct spindle assembly. Indeed, Ase1p phosphorylation coincident with spindle assembly requires Clb5p, Clb3p and Clb4p. Moreover, in clb5Δ cells, Ase1p recruitment and the kinetics of spindle formation were perturbed. These phenotypes were enhanced in a cdc28-4 clb5Δ mutant to the extent that midzone disruption resulted in transient breaks of the short spindle. By contrast, clb3Δ clb4Δ cells delayed spindle assembly downstream to Ase1p recruitment. Expression of Ase1(7D) p that mimics the phosphorylated state restored timely recruitment in clb5Δ cells and fully rescued the corresponding spindle phenotypes. Finally, Ase1(7D) p partially suppressed the spindle assembly delay in clb3Δ clb4Δ cells. Thus, Ase1p phosphorylation by CDK promotes the assembly and stability of the mitotic spindle. It follows that CDK may differentially alter the functionality of members of the Ase1p/PRC1 family to place their distinct roles in their respective stage-specific contexts, a further factor of complexity in the organization of pathways promoting spindle assembly and dynamics.

Membrane topology and post-translational modification of the Saccharomyces cerevisiae essential protein Rot1.

ROT1 is an essential gene that has been related to cell wall biosynthesis, the actin cytoskeleton and protein folding. In order to help to understand its molecular function, we carried out a characterization of the Rot1 protein. It is primarily located at the endoplasmic reticulum-nuclear membrane facing the lumen. Rot1 migrates more slowly than expected, which might suggest post-translational modification. Our results indicate that Rot1 is a protein that is neither GPI-anchored nor O-glycosylated. In contrast, it is N-glycosylated. By a directed mutagenesis of several Asn residues, we identified that the protein is simultaneously glycosylated at N103, N107 and N139. Although the mutation of these three N sites is not lethal, cellular growth is impaired. Sequence analysis predicts a transmembrane domain at the C-terminus. This fragment affects neither the targeting of the Rot1 protein to the ER nor its N-glycosylation, although it is important for the anchoring of the protein to the membrane and for its functionality. The existence of a signal sequence at the N-terminus has been suggested. However, deletion of this fragment impedes neither translocation to the ER nor N-glycosylation, but it is required for cell viability. Finally, we found that Rot1 is translocated to the ER by an SRP-independent post-translational mechanism which depends on Sec62.

Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle.

ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.