Using FASS-LTP in postmortem mice brain tissues to assess pathological synaptic function.

Background: Study of synaptic integrity using conventional electrophysiology is a gold standard for quantitative assessment of neurodegeneration. Fluorescence assisted single-synapse long-term potentiation (FASS-LTP) provides a high throughput method to assess the synaptic integrity of neurotransmission within and between different brain regions as a measure of pharmacological efficacy in translational models.

New Method: We adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts.

Embryonic Tissue and Blastema Transplantations.

Embryo grafts have been an experimental pillar in developmental biology, and particularly, in amphibian biology. Grafts have been essential in constructing fate maps of different cell populations and migratory patterns. Likewise, autografts and allografts in older larvae or adult salamanders have been widely used to disentangle mechanisms of regeneration.

Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration.

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event.

Rebuilding limbs, one cell at a time.

New techniques for visualizing and interrogating single cells hold the key to unlocking the underlying mechanisms of salamander limb regeneration.

The limb enhancer marks an adult subpopulation of injury-responsive dermal fibroblasts.

The heterogeneous properties of dermal cell populations have been posited to contribute toward fibrotic, imperfect wound healing in mammals. Here we characterize an adult population of dermal fibroblasts that maintain an active enhancer which originally marked mesenchymal limb progenitors. In contrast to their abundance in limb development, postnatal enhancer-positive cells (Prrx1) make up a small subset of adult dermal cells (∼0.

Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state.

The journey of cells through regeneration.

The process of building an organ, appendage, or organism requires the precise coordination of cells in space and time. Regeneration of those same tissues adds an additional element of complexity, emerging from the chaos of disease or injury to build a mass of progenitors from mature tissue. Translating insights from natural examples of tissue regeneration into engineered regenerative therapies requires a deep understanding of the journey of a cell directly following injury to its contribution to functional, scaled replacement tissue.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape.

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools.

Connective tissues-skeleton, dermis, pericytes, fascia-are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation.

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array.

XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown.

Regeneration: the ultimate example of wound healing.

The outcome of wound repair in mammals is often characterized by fibrotic scaring. Vertebrates such as zebrafish, frogs, and salamanders not only heal scarlessly, but also can regenerate lost appendages. Decades of study on the process of animal regeneration has produced key insights into the mechanisms of how complex tissue is restored.

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics.

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps).

Using the Drosophila melanogaster D17-c3 cell culture system to study cell motility.

Cultured Drosophila melanogaster S2 and S2R+ cell lines have become important tools for uncovering fundamental aspects of cell biology as well as for gene discovery. Despite their utility, these cell lines are nonmotile and cannot build polarized structures or cell-cell contacts. Here we outline a previously isolated, but uncharacterized, Drosophila cell line named Dm-D17-c3 (or D17).

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration.

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays.

Expression levels of a kinesin-13 microtubule depolymerase modulates the effectiveness of anti-microtubule agents.

Background: Chemotherapeutic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs.

Cortactin promotes cell motility by enhancing lamellipodial persistence.

Background: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration.

Results: Cells deficient in cortactin have impaired cell migration and invasion.