Jay R. Unruh

The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration

Embryonic stem cell–derived fibroblasts with genetic disruption of the Arp2/3 complex are unable to form lamellipodia or undergo sustained directional migration.

Actin Depolymerization Drives Actomyosin Ring Contraction during Budding Yeast Cytokinesis

Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in budding yeast, actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to different ploidies.

Organelle-Based Aggregation and Retention of Damaged Proteins in Asymmetrically Dividing Cells

Aggregation of damaged or misfolded proteins is a protective mechanism against proteotoxic stress, abnormalities of which underlie many aging-related diseases. Here, we show that in asymmetrically dividing yeast cells, aggregation of cytosolic misfolded proteins does not occur spontaneously but requires new polypeptide synthesis and is restricted to the surface of ER, which harbors the majority of active translation sites. Protein aggregates formed on ER are frequently also associated with or are later captured by mitochondria, greatly constraining aggregate mobility. During mitosis, aggregates are tethered to well-anchored maternal mitochondria, whereas mitochondria acquired by the bud are largely free of aggregates. Disruption of aggregate-mitochondria association resulted in increased mobility and leakage of mother-accumulated aggregates into the bud. Cells with advanced replicative age exhibit gradual decline of aggregates-mitochondria association, likely contributing to their diminished ability to rejuvenate through asymmetric cell division.

Cell-Cycle-Coupled Structural Oscillation of Centromeric Nucleosomes in Yeast

The centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast, we developed a method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase-coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell-cycle-coupled oscillation of centromeric nucleosome structure in yeast.

Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes

Meiosis I chromosome migration is biphasic, with an early, slow phase requiring Fmn2 and a later, fast and highly directed phase requiring the Arp2/3 complex.

Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.

Two-photon microscopy with wavelength switchable fiber laser excitation

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

Non-uniform membrane diffusion enables steady-state cell polarization via vesicular trafficking

Actin-based vesicular trafficking of Cdc42, leading to a polarized concentration of the GTPase, has been implicated in cell polarization, but it was recently debated whether this mechanism allows stable maintenance of cell polarity. Here we show that endocytosis and exocytosis are spatially segregated in the polar plasma membrane, with sites of exocytosis correlating with microdomains of higher concentration and slower diffusion of Cdc42 compared with surrounding regions. Numerical simulations using experimentally obtained diffusion coefficients and trafficking geometry revealed that non-uniform membrane diffusion of Cdc42 in fact enables temporally sustained cell polarity. We show further that phosphatidylserine, a phospholipid recently found to be crucial for cell polarity, is enriched in Cdc42 microdomains. Weakening a potential interaction between phosphatidylserine and Cdc42 enhances Cdc42 diffusion in the microdomains but impedes the strength of polarization. These findings demonstrate a critical role for membrane microdomains in vesicular trafficking-mediated cell polarity.

Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors

Arp2/3 complex initiates the growth of branched actin‐filament networks by inducing actin polymerization from the sides of pre‐existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three‐dimensional structures at ∼2 nm resolution of Arp2/3 complex with three different bound NPFs: N‐WASp, Scar‐VCA and cortactin. All of these structures adopt a conformation with the two actin‐related proteins in an actin‐filament‐like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin‐filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.