Biology subjects

Ramirez, A. D.

Publications and source records attributed to Ramirez, A. D..

3 recordsLinked to original sources

bioRxiv2019

Cytoplasmic protein granules organize kinase-mediated RAS signaling

Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner to initiate MAPK signaling. Formation of membraneless protein granules by RTK oncoproteins is both necessary and sufficient for RAS/MAPK signaling output in cells. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform to activate RTKs and RAS GTPases and a general principle by which cells can organize oncogenic signaling. HighlightsO_LIRTK oncoproteins can form de novo membraneless cytoplasmic protein granules C_LIO_LIRTK protein granules activate RAS in lipid membrane-independent manner C_LIO_LIHigher-order protein assembly is critical for oncogenic RAS/MAPK signaling C_LIO_LIProtein granules are a distinct subcellular platform for organizing RTK signaling C_LI

cancer biology↗

bioRxiv2019

Label-retention expansion microscopy

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

bioengineering↗

bioRxiv2018

Pre-saccadic Rise Neurons In The Hindbrain Control The Timing Of Spontaneous Saccades

Organisms have the capacity to make decisions based solely on internal drives. However, it is unclear how neural circuits form decisions in the absence of sensory stimuli. Here we provide a comprehensive map of the activity patterns underlying the generation of saccades made in the absence of visual stimuli. We performed calcium imaging in the larval zebrafish to discover a range of responses surrounding spontaneous saccades, from cells that displayed tonic discharge only during fixations to neurons whose activity rose in advance of saccades by multiple seconds. We lesioned cells in these populations and found that ablation of neurons with pre-saccadic rise delayed saccade initiation. We analyzed spontaneous saccade initiation using a ramp-to-threshold model and were able to predict the times of upcoming saccades using pre-saccadic activity. These findings suggest that ramping of neuronal activity to a bound is a critical component of self-initiated saccadic movements.

neuroscience↗