Cytoskeletal form and function in mammalian oocytes and zygotes.

The actin and microtubule cytoskeletons of mammalian oocytes and zygotes exist in distinct forms at various subcellular locations. This enables each cytoskeletal system to perform vastly different functions in time and space within the same cell. In recent years, key discovery enabling tools including light-sensitive microscopy assays have helped to illuminate cytoskeletal form and function in female reproductive cell biology. New findings include unexpected participation of F-actin in oocyte chromosome segregation, oocyte specific modes of spindle self-organization as well as existence of nuclear actin polymers whose functions are only starting to emerge. Functional actin-microtubule interactions have also been identified as an important feature that supports mammalian embryo development. Other advances have revealed reproductive age-related changes in chromosome structure and dynamics that predispose mammalian eggs to aneuploidy.

The prophase oocyte nucleus is a homeostatic G-actin buffer.

Formation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders and infertility. We found that oocytes contain prominent nuclear F-actin structures that are correlated with meiotic developmental capacity. We demonstrate that nuclear F-actin is a conserved feature of healthy mammalian oocytes and declines significantly with female reproductive ageing. Actin monomers used for nuclear F-actin assembly are sourced from an excess pool in the oocyte cytoplasm. Increasing monomeric G-actin transfer from the cytoplasm to the nucleus or directly enriching the nucleus with monomers led to assembly of stable nuclear F-actin bundles that significantly restrict chromatin mobility. By contrast, reducing G-actin monomer transfer by blocking nuclear import triggered assembly of a dense cytoplasmic F-actin network that is incompatible with healthy oocyte development. Overall, our data suggest that the large oocyte nucleus helps to maintain cytoplasmic F-actin organisation and that defects in this function are linked with reproductive age-related female infertility. This article has an associated First Person interview with Federica Giannini, joint first author of the paper.

Two mechanisms drive pronuclear migration in mouse zygotes.

A new life begins with the unification of the maternal and paternal chromosomes upon fertilization. The parental chromosomes first become enclosed in two separate pronuclei near the surface of the fertilized egg. The mechanisms that then move the pronuclei inwards for their unification are only poorly understood in mammals. Here, we report two mechanisms that act in concert to unite the parental genomes in fertilized mouse eggs. The male pronucleus assembles within the fertilization cone and is rapidly moved inwards by the flattening cone. Rab11a recruits the actin nucleation factors Spire and Formin-2 into the fertilization cone, where they locally nucleate actin and further accelerate the pronucleus inwards. In parallel, a dynamic network of microtubules assembles that slowly moves the male and female pronuclei towards the cell centre in a dynein-dependent manner. Both mechanisms are partially redundant and act in concert to unite the parental pronuclei in the zygote's centre.

Functions of actin in mouse oocytes at a glance.

Gametes undergo a specialized and reductional cell division termed meiosis. Female gametes (oocytes) undergo two rounds of meiosis; the first meiotic division produces the fertilizable egg, while the second meiotic division occurs upon fertilization. Both meiotic divisions are highly asymmetric, producing a large egg and small polar bodies. Actin takes over various essential function during oocyte meiosis, many of which commonly rely on microtubules in mitotic cells. Specifically, the actin network has been linked to long-range vesicle transport, nuclear positioning, spindle migration and anchorage, polar body extrusion and accurate chromosome segregation in mammalian oocytes. In this Cell Science at a Glance article and the accompanying poster, we summarize the many functions of the actin cytoskeleton in oocytes, with a focus on findings from the mouse model system.

Assembly and Positioning of the Oocyte Meiotic Spindle.

Fertilizable eggs develop from diploid precursor cells termed oocytes. Once every menstrual cycle, an oocyte matures into a fertilizable egg in the ovary. To this end, the oocyte eliminates half of its chromosomes into a small cell termed a polar body. The egg is then released into the Fallopian tube, where it can be fertilized. Upon fertilization, the egg completes the second meiotic division, and the mitotic division of the embryo starts. This review highlights recent work that has shed light on the cytoskeletal structures that drive the meiotic divisions of the oocyte in mammals. In particular, we focus on how mammalian oocytes assemble a microtubule spindle in the absence of centrosomes, how they position the spindle in preparation for polar body extrusion, and how the spindle segregates the chromosomes. We primarily focus on mouse oocytes as a model system but also highlight recent insights from human oocytes.

Monitoring SPB biogenesis in fission yeast with high resolution and quantitative fluorescent microscopy.

Like centrosomes, yeast spindle pole bodies (SPBs) undergo a tightly controlled duplication cycle in order to restrict their number to one or two per cell and promote the assembly of a bipolar spindle at mitotic entry. This conservative duplication cycle is tightly coordinated with cell cycle progression although the mechanisms that ensure this coordination remain largely unknown. In this chapter, we describe simple high resolution microscopy- and quantitative light microscopy-based methods that allow to monitor SPB biogenesis in fission yeast and may be useful to study the molecular pathways controlling the successive phases of the duplication cycle.

Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways.

Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors.

Oscillatory AAA+ ATPase Knk1 constitutes a novel morphogenetic pathway in fission yeast.

Cellular morphogenesis relies partly on cell polarization by the cytoskeleton. In the fission yeast Schizosaccharomyces pombe, it is well established that microtubules (MTs) deliver the spatial cue Tea1, a kelch repeat protein, to the tip regions to direct the growth machinery at the cell tips driving the linear extension of the rod-shaped organism to maintain a straight long axis. Here, we report the characterization of Knk1 (kink), a previously unidentified member of the superfamily of ATPases associated with various cellular activities (AAA(+)), whose deletion causes a unique morphological defect characterized by the formation of kinks close to cell tips. Through genetic analysis, we place Knk1 into a novel pathway controlling cell shape independently of MTs and Tea1. Knk1 localizes at cell tips. Its localization is mediated by the Knk1 N terminus and is enhanced upon ATP binding to the C-terminal ATPase domain. Furthermore, Knk1 tip recruitment is regulated by SRC-like adaptor 2 (Sla2) and cell division cycle 42 (Cdc42) independently of Sla2's role in endocytosis. Finally, we discovered that Knk1 shows an anticorrelated oscillatory behavior between the two cell tips at a periodicity that is different from the reported oscillatory Cdc42 dynamics.

Csi1p recruits alp7p/TACC to the spindle pole bodies for bipolar spindle formation.

Accurate chromosome segregation requires timely bipolar spindle formation during mitosis. The transforming acidic coiled-coil (TACC) family proteins and the ch-TOG family proteins are key players in bipolar spindle formation. They form a complex to stabilize spindle microtubules, mainly dependent on their localization to the centrosome (the spindle pole body [SPB] in yeast). The molecular mechanism underlying the targeting of the TACC-ch-TOG complex to the centrosome remains unclear. Here we show that the fission yeast Schizosaccharomyces pombe TACC orthologue alp7p is recruited to the SPB by csi1p. The csi1p-interacting region lies within the conserved TACC domain of alp7p, and the carboxyl-terminal domain of csi1p is responsible for interacting with alp7p. Compromised interaction between csi1p and alp7p impairs the localization of alp7p to the SPB during mitosis, thus delaying bipolar spindle formation and leading to anaphase B lagging chromosomes. Hence our study establishes that csi1p serves as a linking molecule tethering spindle-stabilizing factors to the SPB for promoting bipolar spindle assembly.

Motor proteins: kinesin can replace Myosin.

Directional transport of specific cargos is tuned to specific molecular motors and specific cytoskeletal tracks. Myosin V transports its cargo on actin cables, whereas kinesin or dynein transport their cargo on microtubules. A recent study shows that an engineered kinesin can substitute for myosin V and its cargo-specific transport and subsequent cellular functions.