Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.

In axons, proper localization of proteins, vesicles, organelles, and other cargoes is accomplished by the highly regulated coordination of kinesins and dyneins, molecular motors that bind to cargoes and translocate them along microtubule (MT) tracks. Impairment of axonal transport is implicated in the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Huntington's diseases. To understand how MT-based cargo motility is regulated and to delineate its role in neurodegeneration, it is critical to analyze the detailed dynamics of moving cargoes inside axons. Here, we present KymoAnalyzer, a software tool that facilitates the robust analysis of axonal transport from time-lapse live-imaging sequences. KymoAnalyzer is an open-source software that automatically classifies particle trajectories and systematically calculates velocities, run lengths, pauses, and a wealth of other parameters that are characteristic of motor-based transport. We anticipate that laboratories will easily use this package to unveil previously uncovered intracellular transport details of individually-moving cargoes inside neurons.

Characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate ("map") the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. "Cargo mapping" consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to "map" them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.