Cell Signaling · Unit 5: Signals that build and defend an animal

Notch: Signaling by Contact and by Cutting

Notch is a receptor that signals only between cells that touch, and it does so by being cut: the freed inner part of the receptor travels to the nucleus and switches genes on. This lesson traces the three cuts, weighs what a pathway with no second messenger and no amplification is good for, and shows how two equal neighbours use it to take opposite fates. Afterwards you can predict what happens when the receptor, the ligand or the cutting enzyme is missing or stuck on.

Watch first The Receptor Is the Message · 2:45

A fruit fly with one broken copy of a gene called Notch has nicks in the edges of its wings. An embryo with no working copy builds too much nervous system and too little skin.

The nicked wing, first described in 1914, gave the gene its name. The embryo tells more. In the part of the embryo’s outer layer that gives rise to the nervous system, each cell chooses between becoming a nerve precursor and becoming skin. Normally only some choose nerve. Without Notch, far too many do. So Notch is not what makes a nerve cell. It is what lets a cell be told “not you”.

The fly gene was sequenced in 1985 by the laboratory of Spyros Artavanis-Tsakonas, and soon after by that of Michael Young. It encodes a large protein that crosses the membrane once, with 36 repeats on its outer part that resemble epidermal growth factor. In the same issue of Cell, Iva Greenwald reported the same kind of repeat in lin-12, a worm gene whose mutants make one cell take the fate of its neighbour. A fly and a worm use one family of receptors to settle what two touching cells become. The sequence did not say how. The answer has two parts: contact, and cutting.

The idea: the signal is a touch and a pull

Most signals you have met leave the cell that makes them. Notch’s ligands do not. In flies they are Delta and Serrate; in mammals, the Delta-like and Jagged proteins. Each crosses the membrane of the sending cell and stays anchored there, while the receptor is anchored in the receiving cell. The two can meet only where two cell surfaces meet, so a cell hears Notch only from cells it touches. In one early test, cultured fly cells that do not normally stick together were made to produce either Notch or Delta, and the Notch cells clumped with the Delta cells.

Binding is not enough. In culture, a piece of ligand free in solution generally fails to switch Notch on, while the same piece fixed to a dish works. In the standard account the receptor has to be pulled. After binding, the sending cell draws its ligand back inside by endocytosis, the pinching-in of membrane to form a vesicle, and the tug passes to the receptor it holds.

Just outside the membrane, Notch folds into a compact lock called the negative regulatory region. Three small modules wrap around the spot where the receptor can be cut and hide it from proteases. The pull peels the lock open. At this point the receptor has registered one fact: a neighbour is holding on and pulling.

Three cuts: how the receptor becomes the message

  1. While the receptor is made. In mammalian cells a furin-like protease in the Golgi cuts the new receptor once in its outer part. The two pieces stay clasped together and reach the surface that way. This cut prepares the receptor and carries no signal.
  2. After the pull. An ADAM protease (ADAM10 in mammals) cuts at the newly exposed site, just outside the membrane. The outer part leaves with the ligand, and a stub stays in the membrane.
  3. Inside the membrane. γ-Secretase, a complex of four proteins whose cutting subunit is presenilin, cuts the stub within its membrane-spanning segment. It accepts a membrane protein only after most of the outer part has been trimmed away, which is why the second cut must come first.

The third cut happens in a strange place. Cutting a peptide bond uses water, and the middle of a membrane is oily, so presenilin keeps its two catalytic aspartates in a pocket that water can reach.

That cut frees the inner part of the receptor, the Notch intracellular domain (NICD), which enters the nucleus. A DNA-binding protein called CSL already sits on the target genes and, in the standard account, keeps them off with co-repressors. NICD binds CSL, displaces the co-repressors and recruits a co-activator called Mastermind. The genes turn on. Many encode repressors of the Hes family.

How was it shown that the cut-off inner part is the signal? In steps, each answering an objection to the one before.

In 1993 Gary Struhl, Kevin Fitzgerald and Iva Greenwald made flies and worms that produced only the inner part of Notch or LIN-12, with nothing left to bind a ligand. It changed cell fates as an activated receptor does, and in flies it was in the nucleus. Other fly laboratories reported active truncated Notch that year.

But a fragment made in excess can end up anywhere. In 1996 Raphael Kopan, Eric Schroeter, Harold Weintraub and Jeffrey Nye used a mouse Notch1 that lacked its outer part and was still anchored in the membrane. It was cut, its inner part went to the nucleus, and protease inhibitors stopped the cut.

In 1998 Struhl and A. Adachi tested the intact receptor in a living fly, where too little reaches the nucleus to see by staining. They placed a transcription activator inside Notch, so that a reporter gene turned on only if that part of the receptor reached the nucleus. It did, and only with ligand. Kopan’s laboratory reported ligand-induced release from mouse Notch1 the same year, and two other fly laboratories (Lecourtois and Schweisguth; Kidd, Lieber and Young) reported ligand-dependent processing of Notch.

In 1999 Struhl and Greenwald showed that flies without Presenilin cannot signal through Notch; other laboratories reported the same need in flies and in mouse cells at the same time.

What this design gives up, and what it buys

Set Notch beside a kinase cascade. There, each active enzyme modifies many substrates, so the signal grows at every step, and phosphatases can reset it. Notch has no second messenger and no kinase relay. Between the membrane and the gene nothing multiplies: one cut receptor gives one NICD. A cut cannot be undone, so each receptor signals once.

Set it beside the STAT and Smad routes from Unit 4. There too a protein travels from the membrane to the nucleus and acts on genes. But those receptors are kinases, or borrow one, and one active receptor can phosphorylate one STAT or Smad after another. In Notch the traveller is a piece of the receptor itself.

What the cell buys is a count. The amount of NICD made follows, one for one, the number of receptors a neighbour has pulled and cut. The signal cannot spread to cells that are not touching. It is also brief: a kinase in the nucleus marks NICD’s tail, an E3 ligase adds ubiquitin, and the proteasome destroys it (see the lesson on ubiquitin and proteolysis), so the genes go quiet soon after the cutting stops.

The price is sensitivity to dose. With no amplification to cover a shortfall, half the normal amount of a component can show. That is a likely reason a fly with one working copy of Notch has nicked wings.

Two equal neighbours, two fates

Watch Two Equal Cells, Two Fates · 1:26

In the gonad of a worm larva two cells, Z1.ppp and Z4.aaa, lie side by side. In every worm one becomes the anchor cell and the other a ventral uterine precursor, but which is which varies: each takes the anchor fate in about half of animals. Kill either one with a laser and the survivor always becomes the anchor cell, so the other fate needs a neighbour. Greenwald’s thesis work with Paul Sternberg and Robert Horvitz tied the choice to lin-12. With no LIN-12, both cells become anchor cells. With overactive LIN-12, neither does.

Design problem

Two neighbouring cells start out the same. One must become a nerve cell and the other a skin cell, and nothing outside tells them which. Design a circuit between the two cells that turns a small chance difference into opposite fates. Then predict what the tissue looks like if the receptor in your circuit is missing from every cell.

One way to do it

Give each cell a ligand on its surface and a receptor for that ligand. Add two rules. First, signal received through the receptor blocks the nerve program, so a cell that receives enough becomes skin. Second, the same received signal lowers the cell’s own production of ligand.

Now let chance give cell A slightly more ligand. Cell B receives more signal and makes less ligand. Cell A then receives less signal and makes more. Each turn of the loop widens the gap, until A sends without receiving (nerve) and B receives without sending (skin). Two inhibitions in a row make positive feedback, the switch-making loop from the lesson on signaling circuits, here stretched across two cells.

Without the receptor, no cell can be told “not you”. Every cell runs the nerve program, and the tissue has too many nerve cells and too little skin. That is the fly embryo at the top of this lesson. It is also the worm with no LIN-12, where both cells take the same fate.

Real cells are wired much like this, and the arrangement is called lateral inhibition. In the worm, both cells at first make LIN-12 and its ligand, LAG-2. Before the fates are fixed, the future uterine precursor keeps the receptor and loses the ligand, and the future anchor cell does the reverse. In mosaic worms in which only one of the two cells lacks LIN-12, that cell always becomes the anchor cell: the cell that cannot hear ends up as the sender. In the textbook model of the fly, received signal turns on Hes-type repressors, which shut off the genes that drive both the nerve fate and Delta production. Because the signal neither spreads nor multiplies, each cell compares itself only with the cells it touches, and the pattern comes out fine-grained: one nerve precursor among cells that become skin.

When Notch is stuck on, or missing

Stuck on. In 1991 the human gene NOTCH1 was found broken by a chromosome rearrangement in rare cases of T-cell acute lymphoblastic leukaemia, leaving a shortened receptor. In 2004 mutations in NOTCH1 were reported in more than half of cases. They cluster in two places. Some loosen the lock near the membrane, so the second cut happens without a pull. Others remove the part of the tail that marks NICD for destruction, so each NICD lasts longer.

Missing. Alagille syndrome is a dominant genetic disease. In most cases one copy of JAG1, the gene for the ligand Jagged1, is broken. An affected child has too few bile ducts in the liver and often a heart defect. As in the fly’s wing, one good copy is not enough.

Shared scissors. γ-Secretase also cuts the amyloid precursor protein, and one product is the peptide that builds up in the brain in Alzheimer’s disease. A worm made the connection in 1995, when Diane Levitan and Greenwald found that sel-12, a gene that helps lin-12 signal, resembles a human gene whose mutations cause an inherited, early-onset form of the disease. Drugs that block γ-secretase were developed to lower the peptide, and they block the Notch cut as well. In rodents they turned dividing cells of the gut lining into mucus-secreting goblet cells, the change expected when Notch is lost there. A large trial of one such drug, semagacestat, was stopped in 2010 after patients taking it did worse than those on placebo.

How we measure it

Laser ablation of single cells

A laser focused through a microscope kills one chosen cell in a living, transparent worm. What the remaining cells then do shows what they needed from the cell that is gone. Here it showed that the choice between anchor cell and uterine precursor depends on a neighbour.

Genetic mosaics

An animal is made in which some cells carry a working gene and others do not. Asking which cell must have the gene shows where the gene acts. For lin-12, the cell that lacks the receptor always takes the anchor cell fate, so the receptor acts in the cell that receives.

Truncated receptors

Parts of a receptor are deleted and what remains is produced in cells or animals. If a fragment acts like the activated receptor with no ligand present, that fragment carries the signal. The inner part of Notch passed this test in flies, worms and mouse cells.

Nuclear-access reporter

A transcription activator such as Gal4-VP16 is inserted into the receptor, and a reporter gene responds to it. The reporter turns on only if that part of the receptor reaches the nucleus, which detects amounts too small to see by antibody staining.

Finish line

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Notch1 negative regulatory region — the lock on the second cut
Notch1 negative regulatory region — the lock on the second cut ● LIVE · RCSB PDB 3ETO
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Loading live structure 3ETO from RCSB PDB…

Three small LNR modules, each holding a calcium ion, wrap around the heterodimerization domain and cover the site of the second cut. Picture a pull on the top of the stack lifting them away.

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Notch1 bound to the ligand DLL4 — two membrane proteins meeting between cells
Notch1 bound to the ligand DLL4 — two membrane proteins meeting between cells ● LIVE · RCSB PDB 4XLW
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Loading live structure 4XLW from RCSB PDB…

Two thin rods lie against each other along a narrow strip. In the cell each rod continues to its own membrane, so this strip of contact is what carries the pull from one cell's ligand to the other cell's receptor.

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Notch inner part with CSL and Mastermind on DNA — the switch at the gene
Notch inner part with CSL and Mastermind on DNA — the switch at the gene ● LIVE · RCSB PDB 2F8X
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Loading live structure 2F8X from RCSB PDB…

CSL grips the DNA. The ankyrin repeats of Notch sit on CSL, and one long Mastermind helix lies in the groove the two make together, so Mastermind binds well only when Notch is there.

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γ-Secretase holding a piece of Notch — a cut inside the membrane
γ-Secretase holding a piece of Notch — a cut inside the membrane ● LIVE · RCSB PDB 6IDF
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Loading live structure 6IDF from RCSB PDB…

The membrane-spanning segment of Notch sits inside presenilin, the cutting subunit, with nicastrin, APH-1 and PEN-2 around it. At its inner end the Notch helix turns into a strand that pairs with strands of presenilin, a pairing the cut requires.

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Iva Greenwald

Iva Greenwald

Defined the worm gene lin-12 as a control on the choice between two cell fates, found that it belongs to the same receptor family as fly Notch, and with Gary Struhl showed that the receptor's inner part is active on its own and that Presenilin is needed for Notch to signal.

Greenwald came to Notch through a worm. In her thesis work with Paul Sternberg and Robert Horvitz she defined the gene lin-12 by its mutants, in which one cell takes the fate that normally belongs to its neighbour. In December 1985, as sole author, she reported cloning the gene and finding repeats that resemble epidermal growth factor, the same kind of repeat reported for fly Notch in the same issue of Cell. In 1989 her laboratory, with John Yochem at the bench, and Judith Kimble's laboratory in a companion paper, showed that a second worm gene, glp-1, encodes a similar membrane protein, so two genes known only from their mutants became one receptor family. In 1993, with Gary Struhl and Kevin Fitzgerald, she showed that the inner part of the receptor works without the rest. In 1995, with Diane Levitan, she found that sel-12, a worm gene that helps lin-12 signal, resembles a human gene mutated in early-onset familial Alzheimer's disease. That match was an early sign that Notch signalling and Alzheimer's disease share a protein, found before the enzyme complex itself had been defined.

✓ verbatim Greenwald, Cell 43:583 (1985); Struhl, Fitzgerald & Greenwald, Cell 74:331 (1993).
Gary Struhl

Gary Struhl

Showed in flies that the inner part of Notch alone acts like an activated receptor and is found in the nucleus, then that the intact receptor sends its inner part to the nucleus only when ligand is present, and that this needs Presenilin.

Struhl's Notch papers answer three questions in order. In 1993, with Kevin Fitzgerald and Iva Greenwald, he made flies that produced only the inner part of Notch, with nothing left outside the cell to bind a ligand. Stained with antibody, the protein was in the nucleus, and it changed cell fates as an activated receptor does. That left a fair objection: a fragment made in excess might reach the nucleus for no good reason. In 1998, at Columbia University with A. Adachi, he placed a transcription activator inside the full-length receptor, so that a reporter gene would turn on only if that part of Notch reached the nucleus. The reporter turned on, and only where the ligand was present. In 1999, with Greenwald, he showed that flies lacking Presenilin fail to signal through Notch and that Notch no longer gains access to the nucleus. Together the papers made it reasonable to treat a membrane receptor as a gene regulator kept in storage at the cell surface until a neighbour releases it.

✓ verbatim Struhl & Adachi, Cell 93:649 (1998); Struhl & Greenwald, Nature 398:522 (1999).
Raphael Kopan

Raphael Kopan

Showed with mouse Notch1 that a membrane-anchored receptor lacking its outer part is cut, that its inner part then enters the nucleus, and that protease inhibitors block the cut.

The 1993 experiments used a free inner fragment of Notch, and a sceptic could ask whether anything was really being cut. Kopan, with Eric Schroeter, Harold Weintraub and Jeffrey Nye, answered with mouse Notch1. Their version of the receptor had lost its outer part, so it could not bind a ligand, but it was still anchored in the membrane. In a paper dated 20 February 1996 they reported that this protein was cut and that the freed inner piece went to the nucleus. Protease inhibitors stopped the cut. One inhibitor experiment proves less than it seems, since such compounds often block several proteases, so the result stood as one line of evidence among several. Two years later Kopan's laboratory at Washington University in St. Louis reported, with Schroeter and Kisslinger, that ligand binding causes the same release from the full-length receptor, and identified where the cut falls. That made it possible to detect active Notch directly, with an antibody that recognises only the newly cut end.

✓ verbatim Kopan, Schroeter, Weintraub & Nye, Proc. Natl. Acad. Sci. USA 93:1683 (1996); Schroeter, Kisslinger & Kopan, Nature 393:382 (1998).
Test yourself
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Why does a cell receive a Notch signal only from cells that it touches?
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Every structure, slide, simulation, and clip on this page is listed below with the source it came from. None of the 3D structures is drawn by hand or by AI: each one loads by accession ID from RCSB PDB or PubChem. This panel is generated from the lesson's own data file, so what it lists is what the page loads.

Molecular structures live data

  • Notch1 negative regulatory region — the lock on the second cut — RCSB PDB 3ETO · coordinates CC0 · Human NOTCH1 negative regulatory region, X-ray crystal structure (Gordon et al., Blood, 2009)
  • Notch1 bound to the ligand DLL4 — two membrane proteins meeting between cells — RCSB PDB 4XLW · coordinates CC0 · Notch1 EGF-like repeats 11 to 13 with the receptor-binding end of Delta-like 4, X-ray crystal structure (Luca et al., Science, 2015)
  • Notch inner part with CSL and Mastermind on DNA — the switch at the gene — RCSB PDB 2F8X · coordinates CC0 · Human NOTCH1 ankyrin repeats, CSL and a Mastermind-like 1 peptide on DNA, X-ray crystal structure (Nam et al., Cell, 2006)
  • γ-Secretase holding a piece of Notch — a cut inside the membrane — RCSB PDB 6IDF · coordinates CC0 · Human γ-secretase with a fragment of Notch, cryo-electron microscopy (Yang et al., Nature, 2019)

Scientist profiles & quotes

  • Iva Greenwald — Greenwald, Cell 43:583 (1985); Struhl, Fitzgerald & Greenwald, Cell 74:331 (1993).
  • Gary Struhl — Struhl & Adachi, Cell 93:649 (1998); Struhl & Greenwald, Nature 398:522 (1999).
  • Raphael Kopan — Kopan, Schroeter, Weintraub & Nye, Proc. Natl. Acad. Sci. USA 93:1683 (1996); Schroeter, Kisslinger & Kopan, Nature 393:382 (1998).

Quotes are verbatim from the cited source. Profile text is original to this site and was checked against the sources cited.

Animated explainer

The Receptor Is the Message · narration: an AI narrator voice (Gemini, Charon) reading narration written from this lesson's text · animation: AI-generated in code (Manim) by Claude, a schematic with no drawn molecular structures, plots computed from their equations · captions from the narration script.

Section films

  • Two Equal Cells, Two Fates · narration: an AI narrator voice (Gemini, Charon) reading narration written from this lesson's text · animation AI-generated in code (Manim) by Claude, a schematic with no drawn molecular structures · captions from the narration script.

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Like I'm five

Two friends stand side by side, and each one wants to be the leader. They hold hands and tug. The one who tugs a little harder makes the other tug less, and soon one is the leader and the other is the helper. Cells do this with a protein called Notch: a tug from the neighbour snips a piece off Notch, and that piece carries the message "you are the helper" to the middle of the cell.
High school

Notch is a receptor on a cell's surface, and its signal is a protein fixed on the surface of the cell next door, so only touching cells can use it. When the neighbour's protein binds and pulls, enzymes cut the Notch receptor twice. The second of those cuts frees the part of Notch inside the cell, which travels to the nucleus and switches genes on. Nothing is passed down a chain and nothing is multiplied: one receptor gives one message, once. Cells use this to become different from their neighbours. A cell that receives a lot of Notch signal makes less of the signalling protein itself, so two cells that start equal end up as one sender and one receiver.
College

Notch is a single-pass membrane receptor whose ligands (Delta and Serrate in flies, the Delta-like and Jagged proteins in mammals) are also single-pass membrane proteins, so the signal passes only between cells in contact. The mature mammalian receptor has already been cut once in the Golgi by a furin-like protease. Ligand binding plus a pull from the sending cell, which takes the ligand in by endocytosis, opens the negative regulatory region and exposes a second site, which an ADAM protease cuts. γ-Secretase, whose catalytic subunit is presenilin, then cuts within the membrane and releases the Notch intracellular domain (NICD). NICD enters the nucleus, binds the DNA-binding protein CSL and recruits Mastermind, turning on targets such as the Hes genes. The path has no second messenger and no kinase relay, so nothing is amplified before the gene, and each receptor is used once. In lateral inhibition, received signal lowers a cell's own ligand, so a small difference between neighbours grows into opposite fates.
Graduate

Notch is activated by regulated intramembrane proteolysis. The negative regulatory region (three LNR modules and the heterodimerization domain) buries the S2 site. Crystal structures of this region and single-molecule pulling experiments support a model in which a force of a few piconewtons, generated when the sending cell endocytoses ubiquitinated ligand, exposes S2 to ADAM10. γ-Secretase then cleaves the membrane stub at S3, a step limited mainly by the size of the remaining ectodomain and not by its sequence. NICD assembles with CSL and Mastermind on DNA in place of co-repressors, and phosphorylation of its PEST region followed by ubiquitination by FBXW7 limits its lifetime. Because the stoichiometry is one NICD per activated receptor, output depends on gene dose, consistent with haploinsufficiency for Notch in flies and for JAG1 in Alagille syndrome. NOTCH1 mutations in T-cell leukaemia hit the two controls on this scheme: the heterodimerization domain (S2 cleavage without ligand) and the PEST region (longer NICD lifetime).

An open question is how the first difference between two cells arises and how the feedback is wired in a given tissue. In the worm's anchor cell decision, expression of receptor and ligand diverges before commitment. In fly neuroblast selection, experiments with uniformly expressed Delta suggest that transcriptional control of the ligand is not the only route. Control of ligand activity by endocytosis, or inhibition of Notch by ligand in the same cell, may carry part of the loop.
One cell that kept the others dividing

At the MRC Laboratory of Molecular Biology in Cambridge, Judith Kimble used a laser microbeam to kill a single cell, the distal tip cell, at the end of a living worm's gonad. The germ cells behind it had been dividing. With that one neighbour gone, they stopped and entered meiosis, the division that makes eggs and sperm. No molecule was known. Six years later, with J. Austin, she showed that worms with a broken glp-1 gene behave the same way with the tip cell intact. In 1989 glp-1 proved to encode a relative of Notch.

— Kimble & White, Developmental Biology (1981); Austin & Kimble, Cell (1987).
Sixteen pages apart

In December 1985 one issue of the journal Cell carried two papers sixteen pages apart. The first, from the laboratory of Spyros Artavanis-Tsakonas, gave sequence from fly Notch, a gene known from its mutant since 1914. The second, by Iva Greenwald alone, described the worm gene lin-12. Both proteins contained repeats resembling epidermal growth factor. A gene named for a nick in a fly's wing and a gene found because worm cells swapped fates belonged to one family. Michael Young's laboratory, which had cloned fly Notch independently, published its own sequence soon after.

— Wharton, Johansen, Xu & Artavanis-Tsakonas, Cell 43:567 (1985); Greenwald, Cell 43:583 (1985).
A worm gene that pointed at Alzheimer's disease

In 1995 Diane Levitan and Iva Greenwald were studying sel-12, a worm gene found because mutations in it weaken the effect of an overactive lin-12. Its sequence resembled a human gene, then called S182, whose mutations cause early-onset familial Alzheimer's disease. The result did not come from an Alzheimer's laboratory. Four years later, experiments in flies and mouse cells showed that this protein, now called presenilin, is needed for the cut that frees the inner part of Notch. The same enzyme cuts the amyloid precursor protein, the source of the peptide that builds up in the brain in the disease.

— Levitan & Greenwald, Nature 377:351 (1995); Struhl & Greenwald, Nature 398:522 (1999).

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