Extreme Shock Pressures: Recovery and Detection of Microfossils.

In this thesis an experimental method is utilised to test the viability and suitability of algal microfossils in the context of simulating the shock phase of lithopanspermia. Previously, the lunar surface has been suggested as a potential receptacle and store for ejected terrestrial material following a large impact on Earth. This has led to the moon being labelled in the literature as Earth’s attic. A two stage light-gas gun is used in a series of low velocity and hy pervelocity impacts. These shot range from 0.388 to 5.11 km s-1. These impact velocities experimentally map to computer simulations of ejecta originating from Earth and impacting the lunar surface. Here microfossils are loaded into a sabot and frozen. They are then fired using the light-gas gun at pre-defined velocities at a water bag target. Following the impact the water is filtered and the filtrate analysed under a scanning electron microscope. This thesis finds a shock pressure related size effect in terms of a number of size metrics. Peak shock pressure is calculated using the Planar Impact Approximation. With this, the maximum shock pressure induced by an impact was calculated to be 13.3 GPa. Microfossil fragments were recovered following each shot but intact examples became rarer as the shot velocity was ramped up. This study also provides a solution to a methodological problem arising from evacuation of a light-gas gun, and the consequential evaporation of liquids within a sabot. Thus a projectile design that can contain liquid at low pressure is made available here

A Destruction/Contraction Gradient Coordinates a Persistent and Polarized Global Actin Flow to Control Cell Directionality

Cell motility is hypothesised to be regulated by a step-wise series of events, beginning with leading-edge extension of the membrane. However, it is unclear how rapid leading-edge movements are capable of generating coordinated cell motion. Posterior to the leading edge is the flowing Actin network, which generates cellular propulsive forces. This flow is driven by a combination of Actin polymerisation pushing against the leading-edge, and myosin mediated contraction of the Actin network. Yet, it is unknown how this flow is organised and whether it is involved in controlling cell migration. Through the development of novel computational tools, I show that Actin retrograde flow in developmentally dispersing Drosophila macrophages is highly coherent. Mathematical analysis of Actin flow within macrophages reveals distinct regions of network compression, which are highly persistent in time compared with the leading edge. This data also highlights super-convergent regions within the flow field that represent a sudden transition from retrograde to anterograde Actin motion, whose polarity with respect to the nucleus strongly correlates with cell motion. This unveils a structure and asymmetry to global Actin flow within migrating cells, which I hypothesise is responsible for driving movement through a ‘rear wheel drive’ mechanism of cell migration as opposed to the putative leading-edge mediated ‘front wheel drive’ mechanism of directing cell motion

Persistent and polarised global actin flow is essential for directionality during cell migration

Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence

Heterotypic contact inhibition of locomotion can drive cell sorting between epithelial and mesenchymal cell populations

Interactions between different cell types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesised to control population-wide behaviours during embryogenesis. However, our understanding of the signals that lead to cell-type specific repulsion and the precise capacity of heterotypic CIL responses to drive emergent behaviours is lacking. Using a new model of heterotypic CIL, we show that fibrosarcoma cells, but not fibroblasts, are actively repelled by epithelial cells in culture. We show that knocking down EphB2 or ERK in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. We also examine the population-wide effects when these various cell combinations are allowed to interact in culture. Unlike fibroblasts, fibrosarcoma cells completely segregate from epithelial cells and inhibiting their distinct CIL response by knocking down EphB2 or ERK family proteins also disrupts this emergent sorting behaviour. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations

A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components