Research Projects

Lymphocyte Microvilli:

Contrary to popular opinion, cell surfaces are rarely flat. Instead, they have highly variable topologies, with several types of large protrusions as well as deep invaginations. We are interested in the surface of lymphocytes. When circulating in blood, these cells are covered by finger-like protrusions called microvilli. Upon stimulation with certain chemokines, lymphocytes undergo three major surface changes: 1) they lose their microvilli; 2) they elongate from a spherical to an oblate shape; and 3) they form sheet-like ruffles at one end.

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The consequences of these topological features for lymphocyte function have hardly been studied at all. To date, only one function of lymphocyte microvilli has been proposed: to segregate adhesion receptors used in extravasation (migration of cells from blood to peripheral tissue). During extravasation, lymphocytes must first decelerate from their high velocity in blood. They do this by rolling on the blood vessel wall, making and breaking connections with the vessel wall. Rolling is necessary to decelerate lymphocytes so that they can respond to signals causing them to stop, spread out on the vessel wall, then squeeze between cells on the vessel wall. Cell surface receptor proteins involved in the rolling phase localize to microvilli, where as those mediating the second phase (stopping and spreading) localize between microvilli. The proposed role for microvilli in this process is that, by providing spatial segregation of these two types of receptors, they allow temporal segregation of adhesion events. To date, relatively crude experiments that either ablate microvilli or mis-localize rolling receptors away from microvilli block extravasation, but these experiments are not sufficient to prove the connection.

Our goals on this project are:
1) to determine the mechanisms by which microvilli assemble.
2) To design inhibitors of these mechanisms that cause specific ablation of microvilli.
3) To test whether microvillar ablation blocks rolling.

In addition to helping us understand basic lymphocyte biology, this project could lead to immuno-suppressive or anti-metastatic drugs.

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Progress so far:
We have developed a quantitative assay for evaluating microvilli based on scanning electron microscopy (SEM). Using this assay, we have determined several basic characteristics of microvilli (Majstoravich et al):
1) They are highly dynamic, growing and shrinking on a time scale of seconds to minutes.
2) They are actin-dependent. Actin polymerization inhibitors cause microvillar retraction.

Aims in the next few years:
Our goal now is to figure out microvillar assembly mechanisms. Since they are actin-dependent, we are concentrating on microvillar actin assembly mechanisms. For this, we are taking two approaches:
1) Test known actin assembly factors by suppressing their expression (RNAi).
2) Purify microvilli, identify constituent proteins by mass spec, and test novel candidate assembly factors. We can separate microvilli from the rest of the cell by simply passing cells through a hypodermic needle!

Our two major candidate assembly factors are: formin proteins (see below); and Arp2/3 complex.

For this project, we rely heavily on the following techniques:
1) SEM.
2) Light microscopy (DIC and fluorescence).
3) Cell culture (300.19 pre-B lymphoma cells).
4) Lymphocyte isolation from human blood.
5) Recombinant DNA technology (of course).

Formin Proteins:

Formins are present in all eukaryotic species examined, and many species possess multiple formin genes. As discussed below, their effects on actin polymerization can vary but all act generally to accelerate polymerization. We have identified 15 mammalian formin genes. By RT-PCR, we find that six formins are expressed in lymphocytes. Our interest in formin proteins stems from the possibility that a formin might be responsible for generating microvillar actin filaments in lymphocytes. We want to determine: 1) the biochemical mechanisms by which individual formins influence actin polymerization; and 2) the roles of individual lymphocyte formin proteins in cells.

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Formins have the following sequence characteristics:
1) The formin homology 2 (FH2) domain, spanning about 400 amino acids, is present in all formins. This domain dimerizes and has potent effects on actin polymerization.
2) The formin homology 1 (FH1) domain is of variable length, is proline-rich, and is always N-terminal to the FH2 domain. FH1 can bind the actin monomer binding protein, profilin, and FH1-bound profilin can alter FH2Ős effects on actin polymerization.

Our phylogenetic analysis of FH2 domains shows that the 15 mammalian formins fall into seven distinct groups. Lymphocytes express formins from five of these groups. We are progressively characterizing each of the lymphocyte formins. Thus far, we are examining two such formins, mDia1 and FRL1.

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For biochemical analysis, we use the following techniques:
1) Spectrofluorimetry to measure formin effects on actin polymerization kinetics and protein-protein interactions.
2) Fluorescence microscopy to observe effects of formins on actin filament length and organization.
3) Analytical ultracentrifugation to determine multimeric states and equilibria for each formin.

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Thus far, we have obtained the following biochemical results:
1) mDia1 is a potent actin nucleation factor.
2) FRLa operates by a very different mechanism, it severs pre-formed filaments.
3) Both formins potently inhibit filament barbed end blocking by a protein called capping protein.
4) Both FH2 domains are dimeric.
5) The N-terminus of mDia1 potently inhibits nucleation by the mDia1 FH2 domain (auto-inhibition). A Rho family GTPase, RhoA, can partially relieve this inhibition.

Our future biochemical goals in the next few years are to:
1) determine how mDia1 nucleation is regulated:
  a) What is the mechanism of auto-inhibition?
  b) What is needed in addition to RhoA to obtain full relief of auto-inhibition?
2) determine how FRL1 activity is regulated. Is it auto-inhibited too? If so, what relieves this inhibition.
3) Determine the effects of other lymphocyte formins on actin polymerization. Are they like mDia1 or like FRL1? Or, do they do something completely different?

In addition, we will determine the roles of these formins in lymphocyte biology, using the following techniques:
1) Immunofluorescence microscopy to localize formins in cells.
2) Protein suppression techniques (eg. RNAi) to study the effects of removing this protein from the cell on cell function.
3) Protein interaction techniques (immunoprecipitation, affinity chromatography, two-hybrid analysis) to identify interacting proteins for these formins.

Formins might be acting in the following lymphocyte processes:
1) Microvillar assembly on resting lymphocytes.
2) Cell polarization and ruffle assembly in chemokine-activated lymphocytes.
3) Actin filament rearrangements during T lymphocyte interaction with Antigen Presenting Cells.
4) Extravasation, independent of microvillar assembly.
5) Chemotaxis on solid surfaces.
6) Vesicle transport.