# In situ cryo-EM of protein condensates in the early secretory pathway of neurons

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $238,500

## Abstract

Neurons are highly polarized cells that exhibit distinct moieties that serve to receive (dendrites) and transmit
(axons) electrochemical signals. In a variety of neurodegenerative diseases, loss of neuronal polarity represents
a hallmark of pathogenesis, and coincides with loss of the endomembrane organization in neurons. Despite a
growing inventory of compartment-specific factors and machinery, we lack a fundamental understanding how
neuronal polarity is established and maintained. Specifically, a vast knowledge gap exists with regard to how
bulk routing of cargo is achieved in neurons. Biosynthetic cargo, such as plasma membrane receptors and
channels involved in relaying the electrochemical signals between neurons, must be selectively delivered to
either dendritic or axonal surfaces to support neuron function. Biosynthesis of secretory cargo in neurons occurs
in the endoplasmic reticulum (ER), a compartment that extends throughout dendritic and axonal parts of the
neuron. At nanoscale domains of the ER, the ER exit sites (ERES), cargo is routed via membranous carriers
toward the Golgi apparatus. Golgi acceptor membranes remain near ERES, forming a 300-nm, nearly spherical
interface that exhibits a high extent of long-range order. In addition to a ribbon-like Golgi in the perinuclear region
of the cell soma, recently, additional Golgi elements termed Golgi outposts, or Golgi satellites, have been
detected at the base of dendritic arbors – prompting the view that Golgi outposts could route cargo into the
dendrite, while putatively, somatic Golgi might route cargo into the axon. In the past years, the powerful
framework of biomolecular condensation has gained attention as a mechanism putatively capable of
compartmentalizing cytosol and structuring membrane compartments, with proteins forming dynamic and flexible
networks by the means of a liquid-liquid phase separation (LLPS). Recent evidence points to the ability of multiple
proteins in the early secretory pathway to undergo LLPS – supporting the view that the early secretory pathway
may be structured by self-organizing protein collectives. We will differentiate human induced pluripotent stem
cells (hiPSCs) into mature cortical neurons that are stably transfected with fluorescently tagged ER-Golgi
interface components as molecular ‘beacons’ to enable cryo-correlative light and elecron microscopy (cryo-
CLEM) to guide cryo-focused ion beam (cryo-FIB) milling in conjunction with cryo-electron tomography (cryo-
ET). Our goal is to dissect the molecular architecture of the dendritic and somatic early secretory pathway at
molecular resolution in situ. This will yield an unprecedented opportunity to systematically compare the molecular
composition of individual secretory hubs, and to identify the role of condensates in the organization of these
critical cellular structures while revealing putative adaptations that might enable selective routing of cargo. Our
approach will fill critical knowledge gaps ...

## Key facts

- **NIH application ID:** 10985181
- **Project number:** 1R21DA061421-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Andreas Max Ernst
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $238,500
- **Award type:** 1
- **Project period:** 2024-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10985181

## Citation

> US National Institutes of Health, RePORTER application 10985181, In situ cryo-EM of protein condensates in the early secretory pathway of neurons (1R21DA061421-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10985181. Licensed CC0.

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