Experiments That Distinguish Mechanisms

In 1955 a group in Louvain proposed a new organelle, the lysosome, from numbers in a table of centrifuge fractions, before anyone could say what it looked like.

They broke rat liver, spun it into five fractions, and measured enzymes in every one. One enzyme, acid phosphatase, did not travel with the mitochondria. That table, and the reasoning applied to it, is the subject of this lesson. A cell cannot be watched doing everything at once, so cell biologists take it apart, follow a batch of molecules through it, or remove one part and see what fails. Each approach answers one kind of question and is easily pushed to answer another. Here you will learn which question each one answers.

The idea: a cell is tested by breaking it carefully

Think of a library hit by a flood. You cannot read every book where it fell, but you can sort the wet pages into piles by size, then look for a stamp that tells you which shelf each page came from. Fractionation does this with a cell. The lesson on a measured eukaryotic cell gave you the sizes involved; here they become the basis of separation.

First the tissue is broken gently in a sucrose solution, so that the plasma membrane tears but organelles stay mostly whole. Then comes differential centrifugation: spin at low force and the largest, densest particles (nuclei, unbroken cells) form a pellet, a packed sediment at the bottom of the tube. Spin the liquid above at higher force and smaller particles pellet, and so on. In de Duve’s modified rat-liver scheme, which added a light mitochondrial fraction to the older four-fraction scheme, this gives five fractions: N (nuclear), M (heavy mitochondrial), L (light mitochondrial), P (microsomal, small membrane fragments) and S (soluble, the cytosol). The biochemistry lesson on purification covers centrifugation itself.

Differential spins separate by size and density together, and particle populations overlap, so every pellet is a mixture. Density-gradient centrifugation refines a fraction: in a tube whose sucrose concentration rises toward the bottom, each particle, given long enough, settles where the solution matches its own buoyant density, whatever its size.

The stamp on each page is a marker enzyme, one known to belong to a single compartment. In rat liver the standard markers are cytochrome oxidase for mitochondria, acid phosphatase for lysosomes, and glucose-6-phosphatase for the endoplasmic reticulum (ER), the membrane network where secreted and membrane proteins are made.

Reading a fractionation table

Three quantities do most of the work.

relative specific activity = (% of total enzyme activity in the fraction) / (% of total protein in the fraction)

A value above 1 means the fraction is enriched for that enzyme compared with the starting homogenate. Recovery is simply the percent of total activity found in the fraction. Latency is activity that cannot be measured until a membrane is opened, usually with detergent, because the substrate cannot reach an enzyme sealed inside a compartment.

Worked problem

Here is a made-up data set for rat liver, invented for teaching. Each row gives the percent of the total (homogenate) amount found in each fraction, and every row adds up to 100.

	N	M	L	P	S
Protein	15	20	5	20	40
Cytochrome oxidase	10	70	15	5	0
Acid phosphatase	8	25	45	12	10
Glucose-6-phosphatase	10	10	10	65	5
Unknown enzyme X	9	24	44	13	10

Step 1: relative specific activity. Divide each activity by the protein in the same column.

• Cytochrome oxidase: M is 70/20 = 3.5 and L is 15/5 = 3.0. It peaks in M.
• Acid phosphatase: L is 45/5 = 9.0 and M is 25/20 = 1.25. It peaks in L.
• Glucose-6-phosphatase: P is 65/20 = 3.25. It peaks in P.
• Enzyme X: L is 44/5 = 8.8 and M is 24/20 = 1.2. Its whole profile, fraction by fraction, follows acid phosphatase, not cytochrome oxidase.

Step 2: enrichment, recovery and purity. Fraction L holds 45% of the acid phosphatase in 5% of the protein: a 9-fold enrichment with 45% recovery. More than half the acid phosphatase is elsewhere, so a pure lysosome preparation would be a low-yield one. And L also carries 15% of the cell’s cytochrome oxidase, which is a relative specific activity of 3.0 for mitochondria in the same fraction. L is enriched in lysosomes, not pure.

Enzyme (a row of the table)

Fraction (a column of the table)

RSA = relative specific activity. Where each enzyme peaks (highest RSA)

Rule to keep: relative specific activity = % of the enzyme’s activity in the fraction ÷ % of the protein in the fraction. A fraction rich in one marker can still carry another, so read several markers together. The numbers are the lesson’s made-up teaching data.

Step 3: latency. Suppose that in intact L particles only 10% of X’s activity can be measured, and detergent raises it to 100%. X is 90% latent, so it sits behind a membrane, inside a particle, rather than stuck to the outside.

Conclusion. X is provisionally lysosomal. To confirm it you would want a second lysosomal marker that tracks X on a density gradient, and imaging of the protein in intact cells (see the lesson on microscopy). A shared peak alone can come from two organelles that happen to sediment together.

Pulse and chase: putting events in order

A fractionation table is a snapshot. To learn the order in which a protein visits compartments, you need to label one batch of it and follow that batch. This is a pulse-chase: give cells radioactive amino acid for a short time (the pulse), then flood them with the unlabelled amino acid (the chase), so that only proteins made during the pulse are labelled.

In 1967 James Jamieson and George Palade did this with slices of guinea pig pancreas, whose acinar cells make large amounts of digestive enzymes for export. After a three-minute pulse of carbon-14 leucine, label was in the rough microsomes, the ribosome-studded ER fragments. Smooth microsomes peaked at seven minutes of chase. Later the label reached the zymogen-granule fraction, the storage granules that release enzyme from the cell.

Two cautions came from the same work. First, the granule fraction was half-maximally labelled at 17 minutes, but when Jamieson and Palade examined that pellet by autoradiography, the early label sat largely in condensing vacuoles that sediment with granules. In intact cells, labelled mature granules appeared at the earliest at 57 minutes. A labelled pellet dates the label’s arrival in the pellet, not in the organelle it is named for. Second, in 1968 they added cycloheximide, a drug that blocks protein synthesis, at 0.5 mM during a 37-minute chase. Synthesis fell by up to 98%, yet transport continued at about 80% of control. Newly made protein is not pushed along by the protein made after it, at least over that interval and in that tissue.

What a pulse-chase cannot show is how the protein moves. The order ER, then smooth membranes, then granules fits vesicles budding and fusing, and it also fits compartments that mature into one another. The full secretory story belongs to the lesson on the secretory pathway.

Breaking and fixing: perturbation and rescue

Localization and timing show that a protein is in the right place at the right time. They do not show that it causes anything. For cause you remove the protein and look for a defect, then put it back and see the defect go away.

The tools come from other courses. RNA interference uses a short RNA, delivered as an siRNA or made inside the cell as an shRNA, to guide destruction of a matching mRNA, reducing the protein. CRISPR uses the nuclease Cas9 and a guide RNA to cut a gene, leaving it broken. Both are taught in the lesson on CRISPR and functional genomics, and building rescue constructs in the lesson on recombinant DNA. Drugs are faster still, as the lesson on drugs against a part shows.

Each tool has its own way of misleading.

• RNAi has off-target effects. In HeLa-cell profiling, unintended targets were enriched for 3′ untranslated regions matching positions 2 to 7 of the guide strand. The answer is a second shRNA against a different part of the same mRNA, and above all rescue: a construct whose mRNA carries silent mutations, synonymous codon changes that the shRNA cannot pair with, while the protein stays the same.
• Drugs hit more than their target. OTS167 was described as an inhibitor of the kinase MELK. In 2017 Ann Lin, Jason Sheltzer and colleagues made MELK-null breast cancer clones by CRISPR; the clones grew well in their assays and were still killed by OTS167 (mean GI50 in Cal51 cells, 10 nM with control guides and 11 nM with MELK guides). Another group later found MELK dependency in MDA-MB-231 cells in a low-density colony assay, so whether those cells need MELK depends on the assay.
• Knockouts can be compensated. In selected zebrafish and mouse models, decay of a mutant mRNA switched on related genes, and alleles that made no mRNA at all lacked this response (El-Brolosy and colleagues, 2019). A mild knockout phenotype does not prove the gene unimportant.

The cleanest test of mechanism is a separation-of-function mutant, one that keeps one activity of a protein and loses another. Stephen Royle, Nicholas Bright and Leon Lagnado found in 2005 that depleting clathrin heavy chain in HEK293 cells prolonged mitosis. Clathrin also coats endocytic vesicles, so the defect could have been indirect. Full-length shRNA-resistant clathrin rescued mitosis; the N-terminal fragment did not. In 2006 Royle and Lagnado reported that a shortened construct partly rescued mitosis while failing to support transferrin uptake, the standard readout of clathrin-mediated endocytosis. A protein that cannot do one job but can partly do the other argues that the two jobs are separate.

Design problem

Depleting protein C by shRNA prolongs mitosis in cultured human cells. Model 1: C acts directly on the spindle. Model 2: the defect is indirect, through C’s role in endocytosis. Design experiments that tell the two models apart.

One way to do it

Start by making the phenotype trustworthy. Use a non-targeting control shRNA, and a second shRNA against a different region of C’s mRNA; if both prolong mitosis, an off-target effect is unlikely. Confirm loss of C by immunoblot.

Next, rescue. Express full-length C from a construct with silent mutations in the shRNA target site, alongside an empty-vector control. Mitosis should return to normal. Check by immunoblot that the rescue protein is made at about the normal level, since heavy overexpression can create effects of its own.

Now separate the models. Score every condition for two things: time in mitosis (by live imaging) and endocytosis (transferrin uptake). Then express shRNA-resistant mutants of C, each designed to keep one activity and lose the other, such as a mutant that cannot bind the endocytic adaptors and one that cannot reach the spindle. Model 1 predicts that a mutant that fails at endocytosis can still rescue mitosis. Model 2 predicts that mitosis is rescued only by versions that restore endocytosis, and that blocking endocytosis another way, without touching C, also prolongs mitosis.

Do not decide on localization. Seeing C on the spindle fits Model 1 but does not exclude Model 2; a fragment can reach the spindle and still fail to rescue. This is the logic of Royle’s clathrin experiments: full-length rescue, failed fragments, and a construct that separated the mitotic and endocytic readouts.