Believing is seeing - the deceptive influence of bias in quantitative microscopy.

The visual allure of microscopy makes it an intuitively powerful research tool. Intuition, however, can easily obscure or distort the reality of the information contained in an image. Common cognitive biases, combined with institutional pressures that reward positive research results, can quickly skew a microscopy project towards upholding, rather than rigorously challenging, a hypothesis. The impact of these biases on a variety of research topics is well known. What might be less appreciated are the many forms in which bias can permeate a microscopy experiment. Even well-intentioned researchers are susceptible to bias, which must therefore be actively recognized to be mitigated. Importantly, although image quantification has increasingly become an expectation, ostensibly to confront subtle biases, it is not a guarantee against bias and cannot alone shield an experiment from cognitive distortions. Here, we provide illustrative examples of the insidiously pervasive nature of bias in microscopy experiments - from initial experimental design to image acquisition, analysis and data interpretation. We then provide suggestions that can serve as guard rails against bias.

A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development.

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.