Maddox AS et al. (2012) Methods Cell Biol "High-resolution imaging of cellular processes in Caenorhabditis elegans."

Maddox PS, Maddox AS

[Methods Cell Biol, 2012]

Differential interference contrast (DIC) imaging of Caenorhabditis elegans embryogenesis led to a Nobel Prize in Physiology or Medicine (Sulston et al., 1983) as did the first use of green fluorescent protein (GFP) in a transgenic C. elegans (Chalfie et al., 1994). Given that C. elegans is free living, does not require exceptional environmental control, and is optically clear, live imaging is a powerful tool in for this model system. Combining genetics with high-resolution imaging has continued to make important contributions to many fields. In this chapter, we discuss how certain aspects of high-resolution microscopy are implemented. This is not an exhaustive review of microscopy; it is meant to be a helpful guide and point of reference for some basic concepts in imaging. While these concepts are largely true for all biological imaging, they are chosen as particularly important for C. elegans.

Maddox PS et al. (2012) Methods Enzymol "Imaging the mitotic spindle."

Maddox AS, D'Amours D, Maddox PS, Ratsima H, Ranjan R, Ladouceur AM, Dorn J

[Methods Enzymol, 2012]

The mitotic spindle, due to its striking form, has been imaged for well over 100 years. Composed largely of microtubules and chromosomes, the spindle also contains numerous proteins whose roles include biochemical and biophysical regulation of mitosis. Given the transient, dynamic nature of the spindle, the light microscope continues to be the main tool employed to unlock its mysteries. In this chapter, we will discuss modern light microscopy techniques commonly used for imaging this intricate cellular machine as well as provide examples and protocols. We will also describe some biological preparations and experimental regimes for investigation of the mitotic spindle.

Maddox AS et al. (2007) Dev Cell "Anillin and the septins promote asymmetric ingression of the cytokinetic furrow."

Desai A, Lewellyn L, Maddox AS, Oegema K

[Dev Cell, 2007]

During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.

Maddox AS et al. (2005) Development "Distinct roles for two C. elegans anillins in the gonad and early embryo."

Maddox AS, Oegema K, Habermann B, Desai A

[Development, 2005]

Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.

Amy Maddox et al. (2006) Development & Evolution Meeting "Cortical symmetry breaking promotes asymmetric closure of the contractile ring"

Lindsay Lewellyn, Karen Oegema, Amy Maddox, Arshad Desai

[Development & Evolution Meeting, 2006]

In many large embryos and in polarized cultured mammalian epithelial cells, cytokinesis is asymmetric within the division plane. We have investigated the molecular mechanism of asymmetric cleavage using the C. elegans early embryo. We took advantage of the reproducibility of cell division events in the C. elegans embryo to design quantitative live imaging based assays for contractile ring and furrow dynamics during cytokinesis. We found that, instead of the even ingression of a symmetric furrow encircling the cell equator, a prominent furrow forms on one side of the embryo and sweeps across the division plane toward the opposite side of the embryo. We also show that the contractile ring is structurally anisotropic, with components enriched on the faster-ingressing edge of the contractile ring. However, the anaphase spindle, the source of the signal for contractile ring assembly, is centrally positioned. We show that anillin and the septins, widely conserved structural components of the contractile ring, are required for the anisotropic distribution of structural components around the ring circumference and asymmetric furrow ingression. Anillin and the septins are not required for embryonic cytokinesis, but they become essential in sensitized backgrounds in which the ring is structurally compromised. Septins form heterooligomers that are thought to polymerize to form a plasma membrane associated filament lattice. Anillin can bind directly to F-actin, active myosin, and septin filaments, making it ideally suited to crosslink the membrane associated septin cytoskeleton with the actomyosin network in the contractile ring. We therefore speculate that, following symmetric signaling and initial deposition of contractile ring components, a stochastic symmetry-breaking event is reinforced by a plasma membrane associated scaffold containing anillin and the septins. In this way, the actomyosin network is clustered in one location around the equatorial circumference and the ring constricts asymmetrically.

Amy S Maddox et al. (2005) International Worm Meeting "Distinct roles for two C. elegans anillins in the gonad and the early embryo"

Amy S Maddox, Karen Oegema, Bianca Habermann, Arshad Desai

[International Worm Meeting, 2005]

Anillins are conserved proteins important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, 2 and 3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. In contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical contractile ruffling and pseudocleavage during pronuclear migration, but not for the establishment of polarity. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2 depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.

Maddox PS et al. (2004) Chromosome Res ""Holo"er than thou: chromosome segregation and kinetochore function in C. elegans."

Cheeseman IM, Maddox PS, Oegema K, Desai A

[Chromosome Res, 2004]

Kinetochores are proteinaceous organelles that assemble on centromeric DNA to direct chromosome segregation in all eukaryotes. While many aspects of kinetochore function are conserved, the nature of the chromosomal domain upon which kinetochores assemble varies dramatically between different species. In monocentric eukaryotes, kinetochores assemble on a localized region of each chromosome. In contrast, holocentric species such as the nematode Caenorhabditis elegans have diffuse kinetochores that form along the entire length of their chromosomes. Here, we discuss the nature of chromosome segregation in C. elegans. In addition to reviewing what is known about kinetochore function, chromosome structure, and chromosome movement, we consider the consequences of the specialized holocentric architecture on chromosome segregation.

Maddox PS et al. (2006) Proc Natl Acad Sci U S A "Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay."

Maddox PS, Portier N, Desai A, Oegema K

[Proc Natl Acad Sci U S A, 2006]

Chromosomes condense during mitotic entry to facilitate their segregation. Condensation is typically assayed in fixed preparations, limiting analysis of contributing factors. Here, we describe a quantitative method to monitor condensation kinetics in living cells expressing GFP fused to a core histone. We demonstrate the utility of this method by using it to analyze the molecular requirements for the condensation of holocentric chromosomes during the first division of the Caenorhabditis elegans embryo. In control embryos, the fluorescence intensity distribution for nuclear GFP:histone changes during two distinct time intervals separated by a plateau phase. During the first interval, primary condensation converts diffuse chromatin into discrete linear chromosomes. After the plateau, secondary condensation compacts the curvilinear chromosomes to form shorter bar-shaped structures. We quantitatively compared the consequences on this characteristic profile of depleting the condensin complex, the mitosis-specific histone H3 kinase Aurora B, the centromeric histone CENP-A, and CENP-C, a conserved protein required for kinetochore assembly. Both condensin and CENP-A play critical but distinct roles in primary condensation. In contrast, depletion of CENP-C slows but does not prevent primary condensation. Finally, Aurora B inhibition has no effect on primary condensation, but slightly delays secondary condensation. These results provide insights into the process of condensation, help resolve apparent contradictions from prior studies, and indicate that CENP-A chromatin has an intrinsic role in the condensation of holocentric chromosomes that is independent of its requirement for kinetochore assembly.

Maddox PS et al. (2007) J Cell Biol "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Oegema K, Monen J, Desai A, Maddox PS, Hyndman F

[J Cell Biol, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Francie Hyndman, Joost Monen, Paul Maddox, Arshad Desai, Karen Oegema

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.