In 2019, over 220,000 Canadians will be diagnosed with cancer and 82,000 will die of the disease. Approximately 90% of cancer related deaths are the result of metastatic disease. This is indeed the case for breast cancer where metastasis is the deadliest aspect of disease progression. We have implicated the Lipoma Preferred Partner (LPP) protein as an important mechanosensitive integrator of TGFβ-mediated breast cancer cell migration and invasion, two cellular processes that are critical for cancer metastasis. Two important structures that regulate migration and invasion are cellular adhesions (migration) and invadopodia (invasion). Cell-matrix adhesions are dynamic structures that link the extracellular matrix to the internal cytoskeleton. Adhesions are tightly regulated in space and time to control cell migration. In turn, invadopodia are structures that form enabling cells in exit primary tumors, enter the blood stream and enter distant tissues at sites of metastasis. Our work shows that LPP is an important regulator of cell-matrix adhesion and invadopodia formation and substrate degradation in response to TGFβ stimulation. We demonstrate this in several cell breast cancer cell lines including triple negative 4T1 cells. Using biophysical tools and quantitative imaging we link LPP to regulation of cell migration through regulation of adhesion dynamics, mechanosensing, regulation of tension at individual adhesions and invasion on substrates with variable biomechanical properties.

Understanding morphogenesis is an inherently four-dimensional problem, as cells change position in space and the embryo is transformed over time. Dorsal intercalation of epidermal cells in the C. elegans embryo is a powerful model for studying how “hybrid” epithelial cells engage in the directional rearrangement known as convergent extension. Dorsal intercalation relies on a conserved Rac/RhoG cassette activated by the Rho family guanine nucleotide exchange factors (GEFs) and negatively regulated by the highly conserved actin capping protein regulator, CRML-1/CARMIL Ongoing work in the lab show intercalation is modulated by the Slit/Robo/srGAP SRGP-1 and the worm homologues of the planar cell polarity components Vangl and Prickle. Multimodal live imaging has been crucial to analyzing dorsal intercalation, and continues to be a key tool for analyzing intercalation at increasing resolution. These studies establish dorsal intercalation as a powerful model system for analyzing convergent extension at single-cell resolution.

Similar to the “histone code” hypothesis in which epigenetic marks on histone tails expand the information potential of the genetic code, different tubulin isoforms, combined with a panoply of post-translational modifications (PTMs), constitute the so-called “tubulin code”. The tubulin code was proposed to account for subcellular differentiation of distinct microtubule populations that control the activity of several molecular motors. One of these PTMs consists in the catalytic removal of the c-terminal tyrosine of most mammalian α-tubulin isoforms by tubulin carboxypeptidases (TCPs), such as the recently identified Vasohibin-SVBP complexes, followed by re-tyrosination of soluble α-tubulin by tubulin tyrosine ligase (TTL). α-tubulin detyrosination has been recently implicated in mitosis and meiosis, neuronal processes and cognitive brain function, heart and skeletal muscle contraction, and cancer. In this seminar, I will discuss our work aiming to elucidate how the tubulin code regulates different aspects behind faithful chromosome segregation during mitosis and the implications for human cancers. In particular, I will focus on recent evidence that a-tubulin detyrosination works simultaneously as a navigation system and as a “mitotic error code” to ensure mitotic fidelity.

Madin Darby Canine Kidney (MDCK) cells can form a polarized epithelial layer in culture. We and others have observed that these layers are frequently characterized by extralayer cells, positioned above (apical to) the layer. One possible origin for extralayer cells is an initial failure in monolayer formation, meaning that cells “clumped” on top of each other during plating. Another possibility is that newly born division products fail to integrate into the extant layer.
To investigate the aetiology of misplaced cells, we have developed an analysis pipeline, including a suite of custom algorithms called Automated Layer Analysis (ALAn), to quantify aspects of MDCK cell layer architecture at different areas, ages, and cell densities. Using ALAn, we have determined that the number of extralayer cells correlates positively with layer density in a non-linear fashion. In addition, the layer becomes more organized over time, with cells settling into a layer by 24 hours, suggesting that these extra-layer cells are not a result of clumping during plating. We are now using this tool to investigate whether extralayer cells are a result of division, and whether or not misplaced cells can reintegrate into the developing monolayer to maintain tissue integrity.
 

During organismal development, differential regulation of the cell cycle is critical to many cell biological processes, including cell migration, cell fate specification and differentiation. While the mechanisms of cell cycle regulation are well studied, how control of the cell cycle is linked to differentiated cellular behavior remains poorly understood, mostly due to our inability to directly and precisely measure cell cycle state. In order to characterize cell cycle state live, we have adapted a cyclin dependent kinase (CDK) biosensor for in vivo use. We demonstrate here using a combination of spinning disk confocal and adaptive optics lattice light sheet microscopy the use of this biosensor to quantify lineage-specific differences between cycling cells, and to examine the link between quiescence and differentiation in the roundworm nematode, Caenorhabditis elegans and zebrafish. Unlike other live cell imaging tools (e.g., FUCCI), we show that DHB can be used to distinguish between actively cycling cells in the G1 phase of the cell cycle and quiescent or terminally differentiated cells exited in G0. Additionally, we have used this tool to investigate cell cycle regulation during morphogenesis and regeneration in zebrafish and through a multi-lab collaboration are exploring the use of this biosensor across the animal kingdom. Thus, we provide here a new resource to study the control and timing of the cell cycle during cell migration, fate specification and differentiation.

Rebecca Adikes1, Nicholas Palmisano1, Abraham Khorman1, Michael Martinez1, Jayson Smith1, Taylor Medwig-Kinney1, Tian-Ming Fu3, Maria Jose Gacha Garay1, Ononnah Ahmed1, Nicholas Weeks1, Nuri Kim1, Simeiyun Liu1, Rob Morabito1, Wan Zhang1, Eric Betzig3, Ariel Pani2, Benjamin Martin1, David Matus1
1) Stony Brook University, Stony Brook, NY 2) University of Virginia, Charlottesville, VA 3) Janelia Research Campus, Ashburn, VA

Although much is known about the molecular maternal-fetal interactions during implantation, the 3D architecture of the uterine environment in which the early embryo develops is not well understood. Using confocal imaging in combination with 3D analysis we identified dynamic changes in the luminal and glandular structure of the murine uterus in preparation for implantation. Further, we used this methodology to uncover patterns and mechanisms of pre-implantation embryo movement in the uterus. Our analysis reveals three distinct pre-implantation stages: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that the unidirectional movement of embryo clusters is facilitated by a mechanical stimulus of the embryo as a physical object and is regulated by adrenergic uterine smooth muscle contractions. Surprisingly, we find that embryo scattering, is independent of muscle contractions but instead relies on LPAR3 signaling mediated embryo-uterine communication. Our data supports a model where murine uterine implantation sites are neither random nor predetermined and are a function of the number of embryos entering the uterine lumen. We propose that the presence of embryo clusters in the uterine horn provides an opportunity for the uterus to sense and count the embryos, followed by scattering and even spacing of these embryos along the given length of the horn. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy.

LED illumination has become more prominent in microscopy for both fluorescence and transmitted applications over the past few years. Today, LEDs provide many benefits over mercury and metal halide lamps that have typically been used by microscopists in the past. The historical shortcomings of LEDs have now been overcome, meaning they are now the most popular illumination devices for microscopy.

This presentation will highlight how recent developments in LEDs have made them the go-to option for lab managers, and explore the benefits of LEDs over mercury lamps for both fluorescence and transmitted work in microscopy. We will explain how they can provide a cost-effective solution, replacing several components required when using mercury. Using data examples, we will also show how using LEDs are preferential for imaging, providing better signal to noise as well as improved cell viability and reduced photobleaching.

Microtubules (MTs) are dynamic cytoskeleton polymers forming a polarized intracellular network that provides structural support, facilitates intracellular transport and drives chromosome segregation. To investigate how microtubules control cell dynamics with high spatial and temporal accuracy, we developed a photo-inactivated variant of the key MT plus-end adaptor EB1, that allows local and acute manipulation of MT growth dynamics by controlling the recruitment of EB-dependent plus end tracking proteins (+TIPs) to MT ends. Blue light-induced rapid and reversible dissociation of the +TIP complex from MTs results in a MT growth rate decrease and induces depolymerization of cell edge associated MTs (van Haren et al. 2018, Nat. Cell Biol. 20, 252–261). I will discuss our development and validation of this optogenetic tool and results supporting independent roles of +TIP complexes and the canonical MT polymerase XMAP215/CKAP5 in promoting physiological MT growth rates in cells, as well as ongoing work investigating the consequences of local modulation of MT growth on neuron morphogenesis.

The transport properties of biomolecules in cells can reveal a great deal about the functional interactions regulating cells at the molecular level. Various biophysical methods have been developed to measure these properties in cells, although most have relied on fluorescence microscopy imaging as the window for measurement of labeled macromolecules in living cells. Image correlation methods are an extension of fluorescence fluctuation spectroscopy that can measure protein-protein interactions and macromolecular transport properties from input fluorescence microscopy images of living cells. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series using a fluorescence or super-resolution microscope. I will introduce spatio-temporal image correlation spectroscopy (STICS) and its 2 color cross-correlation variant (STICCS) and show how the analysis can reveal hidden coupling between retrograde cellular actin flows and the plasma membrane lipids for activated Jurkat cells. I will then describe the application of the STICS and pair vector correlation for measuring cellular waves of adhesion related macromolecules talin and vinculin as well as cytoskeletal actin between assembling and disassembling podosomes in dendritic immune cells. Podosomes are cylindrical membrane complexes with an integrin adhesive ring and an actin rich core that are associated with cellular migration and invasion in specific cell types. EM and super-resolution microscopy of cells shows radial actin filaments that connect neighboring podosomes. The image correlation analysis combined with pharmacological perturbation experiments show that podosome turnover is coordinated within local clusters in cells with a correlation length scale extending to next nearest neighbor podosomes. Moreover, recent work pairing the analysis with live cell super-resolution microscopy reveals that the dynamic coordination between podosomes differs for cells on soft versus stiff substrates which provides clues to the mechanistic function of podosomes.

Multi-colour electron microscopy (MCEM) combines ultrastructural electron data with elemental information, transforming traditionally grey scale electron micrographs into colourful images that combine structure and composition. Energy dispersive x-ray spectrometry (EDS) is a rapid and relatively easy method for determining elemental distribution by measuring the energy of characteristic x-rays emitted from a sample when exposed to an electron beam. Recent key developments in EDS technology have increased the potential for accurate analysis of light elements in biological samples. This tech talk will discuss how our Ultim Extreme EDS detector is optimised for life science applications, ranging from the identification and mapping of endogenous, native elements to the characterisation of implant materials, pollution particles and the interaction of cells and tissues with biomaterials.

Almost unidimensional in their primordial existence as single cells, multicellular organisms quickly develop into complex 3D architectures and machineries composed of multiple cells and tissues. For more than a century, classical microscopy has given us important insights into the mechanics and mechanisms that bring about complex biological forms during their development, but it was only recently that we have been able actually witness those processes it their full complexity and wonder. This was due mainly to the emergence of 3D optical microscopy techniques, such as confocal or two-photon, and more recently of mesoscopic imaging such as light-sheet and optical tomography. I will quickly review some of the principles behind optical tomography/slicing and its use in cell and developmental biology and focus on optical projection tomography (OPT) as a tool for embryologists used to characterize vertebrate embryonic and fetal anatomy with little efforts and great fidelity.

Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins—Plk4, Ana2/STIL, Sas-6, Sas- 4/CPAP or Asl/Cep152—leads to the absence of centrioles. Here we use Sas- 6/Ana2 Particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs/embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, while Asl helps to initiate SAP assembly, but is not required for SAP growth. Although not centrioles, SAPs recruit and organize many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl/Cep152 to efficiently recruit pericentriolar material (PCM), but Spd-2/Cep192 can promote some PCM assembly independently of Asl/Cep152. These observations provide new insights into the pathways of centriole and centrosome assembly.

How organisms control organ size is a fundamental question in developmental and regenerative biology. The underlaying mechanisms are not well defined. For instance, we don’t fully understand how cell proliferation and cell death are mechanistically integrated within tissues to generate stereotypic size and shape characteristics.
Using the developing Drosophila wing, we have identified the JNK interacting protein-3 (JIP3) as a size regulator. Mechanistic studies revealed that JIP3 acts downstream of Yorkie, a broadly conserved tissue size control molecule. Interestingly, JIP3 appears to play a dual role during wing development: one the one hand, JIP3 stabilizes Diap1, a cell survival and Yorkie target molecule. This provides robustness to Yorkie’s growth promoting program during the rapid tissue expansion phase. On the other hand, JIP3 transcription is developmentally downregulated later in development, promoting Diap1 rapid degradation and allowing cell death to occur. This provides a developmentally timed mechanism for counteracting Yorkie-mediated tissue growth.

Confocal microscopes (and related instruments such as Two-Photon) are often used for the most rigorous quantitative fluorescence microscopy measurements. Most confocal users are aware of the basic requirements for generating quantitative images: prepare the samples the same way, image them with the same settings, avoid cross-talk and photobleaching, etc. However, many are unaware of the more subtle (but relatively common) pitfalls to obtaining reproducible results. Here I highlight the hazards and suggest some ways of ensuring rigor and reproducibility in confocal fluorescence microscopy experiments, as detailed in our recent Nature Protocols tutorial paper entitled, “Tutorial: guidance for quantitative confocal microscopy”:
https://www.nature.com/articles/s41596-020-0313-9

The mammary gland is a unique organ that develops predominantly after birth and undergoes dramatic remodelling in reproductive phases. Macrophages have been implicated in mammary gland function and remodelling but their diversity has not been fully addressed. 3D microscopy of optically cleared tissue is a powerful tool for revealing cellular interaction in situ. Using this technique, we have identified a rare and unique population of mammary gland-resident ductal macrophages (DMs). These are dendritic-shaped and form a contiguous network between the luminal and basal layers of the entire mammary gland throughout post-natal development. To directly observe DM behaviour, we developed an intravital imaging approach that allowed high-resolution, 3D imaging of virgin mammary ducts in vivo, revealing the nature of DM–epithelium interactions. Functionally, DMs proliferate in pregnancy to maintain complete coverage of the epithelium in lactation, where they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. In the context of breast cancer, DMs strongly resemble mammary tumour-associated macrophages and increase throughout tumorigenesis to form a network that pervades the tumour epithelium. Thus, the mammary epithelium programs specialised resident macrophages in both physiological and tumorigenic contexts.