Lecture 3: Cell fusion - Heterokaryons. Feb.3, 2000
Heterokaryons
Fusogenic agents: PEG, Sendai virus (syncytia promoting, as HIV).
Heterokaryons vs. Hybrids.
Variations: Cytoplasts (cytochalasin enucleated cells), karyoplasts, reconstructed
cells, mitochondrial inheritance [CAPR, valinomycinR])
Microcells (via colcemid-induced micronuclei + cytochalasin) and fusion.
See later for mapping)
Uses:
Track mobility of molecules and cell structures
Examine trans effects on cell cycle
Examine trans effects on gene regulation (differentiation)
Complementation analysis to define separate genes
Inversitagte natural cell fusio venets (including virus entry)
Mobility:
Edidin: plasma membrane proteins motility
Mitochondria are mobile in fusing myocytes (unlike territoriality of myosin
heavy chain around individual nuclei)
Nuclear-cytoplasmic shuttling proteins (paper for next time)
Gene regulation studies:
Reactivation of pycnotic nuclei (Henry Harris, hen erthyrocytes x HeLa)
RBC=negligible cytoplasm; nucleus swells; Hu nuc. Ag; nucleoli enlarge,
syn RNA; chick products=surface Ag; HPRT; DNA synthesis (H. Harris)
More specific differentiation studies:
Activation of specialized genes (muscle genes in non-muscle cells - paper to be discussed next time)
Even more specific gene regulation studies: reporters to study specific
transcriptional activators :
Transient fusion for biochemical analysis (FB x GH3 -> PRL-CAT in fibroblasts)
(paper to be discussed next time)
Complementation:
Xeroderma pigmentosum unscheduled DNA syn. in DNA repair mutants
Fuse, UV-insult DNA, mutnats do not carry out unscheduled DNA synthesis (repair
synthesis). In heterokaryons, WT nucleus fixes mutant nucleus, ifmutant fixes
mutant nucleus: complementation, therefore mutants are in different repair
genes. I f not fix, then in same gene. Around 9 complementation groups
(genes) for Xp.
HIV viral fusion simulation:
CD4 = viral receptor; gp120, gp41 = viral envelope surface glycoproteins
CD4 in a mouse cell + GP120/GP41 in a CHO cell. Test
therapeutic reagents (e.g., various sCD4 competitor proteins: combo Ig-CD4
current best candidate, slow clearance, high affinity)