Research

Project Overview

The cell comprises a multitude of molecular assemblies residing in specific compartments and active at specific times. Their location and timing are critical. Cancer, for example, results from molecules that are expressed at the wrong level, misplaced, or active all the time.

Somehow all of these assemblies work together to generate functioning cells—the question is, how?

Our first major project will seek to understand the organization and morphologies of the major intracellular structures and regulatory complexes in undifferentiated stem cells and cardiomyocytes derived from them. To do this, we will add fluorescent markers to genome-edited cell lines and use light microscopy to determine the numbers, locations, and dynamic changes of these intracellular structures and regulatory complexes. The data will be used to develop algorithms that predict positions of the structures within the cell, based on the distribution of other structures, and how cell components reorganize as the stem cell goes through the cell cycle and differentiates. These data in turn will be used to characterize and predict cell types and states as well as the effects of altered environments and mutations that cause disease.

Our efforts will culminate in the Allen Cell Explorer—an online image database with tools to visualize, interrogate and annotate dynamic 3D images, cellular models, and other computational outputs.

Allen Cell Collection

The Allen Institute for Cell Science has released the Allen Cell Collection: the first publicly available collection of gene edited, fluorescently tagged human induced pluripotent stem cells that target key cellular structures with unprecedented clarity.

Gene list and status
Updated February 2017

Videos

Gene Editing versus Transient Transfection

Updated June 2016 | View videos of our gene edited cells, comparing the results with traditional transient transfection methods. Each video is a series of slices taken from the bottom of the cells upward, in order to visualize them in three dimensions. The two techniques are presented side by side, with transient transfection on the left and gene editing on the right.

Desmosomes (Desmoplakin) -- Desmosomes function to maintain the structural integrity at adjacent cell contacts, particularly striking in epithelial cells and cardiac muscle. 

Mitochondria (Tom20) -- Mitochondria are double membrane-bound organelles within a cell that generate most of the cell's supply of energy.

Microtubules (Tubulin) -- Microtubules are found throughout the cytoplasm as a component of the cell’s internal skeleton (cytoskeleton). They are important in a number of cellular processes and their role in chromosome separation at mitosis is particularly obvious in the three cells in the top right of the gene edited video.

Z-stacks

Updated February 2017 | View videos of gene edited human induced pluripotent stem cell lines expressing fluorescence tagged markers for the golgi, cytoskeleton, endoplasmic reticulum and nucleolus. Each video is a series of slices taken from the bottom of the cells upward, in order to visualize them in three dimensions. 

Golgi (Beta-galactoside alpha-2,6-sialyltransferase 1) — The Golgi apparatus is organized into folded membranes and vesicles located primarily near the top of undifferentiated stem cells. It is important for intracellular trafficking, especially protein secretion, and also contributes to organizing the microtubule cytoskeleton.

Actin (beta-actin) — Actin is a component of the cell’s internal skeleton (cytoskeleton) and is found in filament bundles that can be seen by light microscopy as linear structures in the cytoplasm as. It plays a critical role in many phenomena including cell migration and division.

Endoplasmic reticulum (Sec61b) — The endoplasmic reticulum is a large membrane-bound organelle that occupies much of the cell. It is a central hub for protein production and membrane trafficking. 

Nucleolus (Fibrillarin) — The nucleolus is a subcompartment of the nucleus where a key cellular machine, the ribosome, is assembled.

Time-lapse Videos

Updated February 2017 | View timelapse videos of our gene edited cell lines.

Mitochondria (Tom20) - Mitochondria are double membrane-bound organelles within a cell that generate most of the cell’s supply of energy. They are very dynamic, moving through the cell, dividing, and fusing with each other. This video shows mitochondria near the bottom of the cell, which was imaged in 3D. Each frame represents 30 seconds and the entire video lasted 30 minutes. These images were collected on the Zeiss Airyscan FAST system in high-resolution mode.

Nuclear envelope (Lamin B1) — This protein is a component of the envelope that surrounds the nucleus. It breaks down and then reforms during cell division, as seen in the two dividing cells in the video. The video shows a single slice near the center of the cells, which were imaged in 3D. Each frame represents 35 seconds and the entire video represents 23 minutes. The video is sped up 350-fold over real time.

Microtubules (alpha tubulin) — Microtubules give structure and enable mobility in cells and are key players in the cell's life cycle and behavior, including in mitosis, intracelluar transport and the cytoskeleton. This timelapse movie shows a hiPSC colony expressing alpha tubulin-mEGFP. Images taken as a single slice near the top of the cell every 1 minute for 65 minutes on a spinning-disk confocal microscope. Movie sped up 900x over real time.

Updated February 2017 | View two timelapse videos of a colony of our gene edited cell lines over many hours. These videos highlight the dynamic nature of these tightly packed cells growing, dividing, and pushing on each other.

Microtubules (alpha-tubulin) - This video shows a maximum intensity projection of a 3D movie. Each frame represents 4 minutes and the entire video lasted 6hours 40 minutes. The video is sped up 1200-fold over real time.

Tight Junctions (tight junction protein ZO-1) - Tight junctions are located at cell-cell interfaces  near the top of our cells, where they help hold the membranes of two cells very tightly together. This video shows a maximum intensity projection of a 3D movie. Each frame represents 3 minutes and the entire video lasted 15 hours. The video is sped up 1800-fold over real time

Z-stacks with Dye

Updated September 2016 | Each video comprises a series of slices taken from the bottom of the cells upward, in order to visualize them in three dimensions. Both of these 3-colored videos show a cellular structure (microtubules or mitochondria) in white, along with two fluorescent dyes that highlight the cell periphery (magenta) and the DNA in the nucleus (cyan). These cells grow in tightly packed colonies and display a stereotyped organization from bottom to top. At the bottom, cells extend thin sheetlike “feet,” sometimes overlapping each other. Moving toward the top of the cells, their shape is columnar with the nucleus taking up most of the central region (~50% of the total volume of the cell). Most of the organelles sit above the nucleus, near the top of the cell, which can lean sideways onto another cell, forming a sort of cyoplasmic “pocket” extension. This pocket region contains the centrosomes, Golgi and densest regions of the microtubule array. Near the top, the cells are tightly connected to their neighbors via cell-cell junctions.