Robert A. Bloodgood

Chapter 5 Targeting Proteins to the Ciliary Membrane

Most vertebrate cell types display solitary nonmotile cilia on their surface that serve as cellular antennae to sense the extracellular environment. These organelles play key roles in the development of mammals by coordinating the actions of a single cell with events occurring around them. Severe defects in cilia lead to midgestational lethality in mice while more subtle defects lead to pathology in most organs of the body. These pathologies range from cystic diseases of the kidney, liver, and pancreas, to retinal degeneration, to bone and skeletal defects, hydrocephaly, and obesity. The sensory functions of cilia rely on proteins localized specifically to the ciliary membrane. Even though the ciliary membrane is a subdomain of the plasma membrane and is continuous with the plasma membrane, cells have the ability to specifically localize proteins to this domain. In this chapter, we will review what is currently known about the structure and function of the ciliary membrane. We will further discuss ongoing work to understand how the ciliary membrane is assembled and maintained, and discuss protein machinery that is thought to play a role in sorting or trafficking proteins to the ciliary membrane.

Sensory reception is an attribute of both primary cilia and motile cilia

A recent cluster of papers has shown that motile cilia in the respiratory and reproductive tracts of humans and other mammals can exhibit sensory functions, a function previously attributed primarily to non-motile primary cilia. This leads to a new paradigm that all cilia and flagella (both motile and primary) can mediate sensory functions. However, examination of the literature shows that evidence of sensory functions of motile cilia and flagella is widespread in studies of invertebrates, and extends as back as far as 1899. In this Opinion article, I review the recent and historical findings that motile cilia have a variety of sensory functions, and discuss how this concept has in fact been evolving for the past century.

From Central to Rudimentary to Primary: The History of an Underappreciated Organelle Whose Time Has Come.The Primary Cilium

For the first time, the history of the central flagellum/primary cilium has been explored systematically and in depth. It is a long and informative story about the course of scientific discovery, memory loss and rediscovery. The progress of our story is saltatory, pushed onward by innovations in technology and retarded by socio-scientific issues of linguistic and temporal chauvinism. Over one hundred and fifty years passed between the discovery of this organelle and full appreciation of its important functions. The main character in our story is an organelle that was relegated to a very minor role in the cellular opera for a very long time, until its rather sudden promotion to a central role in orchestrating many of the sensory and signaling events of the cell. Although early investigators speculated on just such a role for the primary cilium as early as 1898, it was over one hundred years before proof for this hypothesis was forthcoming.

Flagella-dependent gliding motility inChlamydomonas

SummaryFlagella are generally recognized as organelles of motility responsible for the ability ofChlamydomonas to swim through its environment. However, the same flagella are also responsible for an alternative form of whole cell locomotion, termed gliding. Use of paralyzed flagella mutants demonstrates that gliding is independent of axonemal bend propagation. Gliding motility results from an interaction of the flagellar surface with a solid substrate. Gliding is characterized by bidirectional movements at 1.6±0.3 μm/second and occurs when the cell is in a characteristic gliding configuration, where the two flagella are oriented at 180° to one another. A variety of observations suggest that the leading flagellum is responsible for the force transduction resulting in cell locomotion, although both flagella have the capacity to function as the active flagellum. The characteristics of gliding motility have been compared with theChlamydomonas flagellar surface motility phenomenon defined as surface translocation of polystyrene microspheres.

The reciprocal coordination and mechanics of molecular motors in living cells

Molecular motors in living cells are involved in whole-cell locomotion, contractility, developmental shape changes, and organelle movement and positioning. Whether motors of different directionality are functionally coordinated in cells or operate in a semirandom “tug of war” is unclear. We show here that anterograde and retrograde microtubule-based motors in the flagella of Chlamydomonas are regulated such that only motors of a common directionality are engaged at any single time. A laser trap was used to position microspheres on the plasma membrane of immobilized paralyzed Chlamydomonas flagella. The anterograde and retrograde movements of the microsphere were measured with nanometer resolution as microtubule-based motors engaged the transmembrane protein FMG-1. An average of 10 motors acted to move the microsphere in either direction. Reversal of direction during a transport event was uncommon, and quiescent periods separated every transport event, suggesting the coordinated and exclusive action of only a single motor type. After a jump to 32 °C, temperature-sensitive mutants of kinesin-2 (fla10) showed exclusively retrograde transport events, driven by 7 motors on average. These data suggest that molecular motors in living cells can be reciprocally coordinated to engage simultaneously in large numbers and for exclusive transport in a single direction, even when a mixed population of motors is present. This offers a unique model for studying the mechanics, regulation, and directional coordination of molecular motors in a living intracellular environment.

Directed movements of ciliary and flagellar membrane components: A review

Summary— The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chalmyodomonas flagellum, where surface motility has been most extensively studied, it appears that cross‐linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane‐matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Clamydomonas gametes, which is initiated by flagellum‐flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.

Active learning: A small group histology laboratory exercise in a whole class setting utilizing virtual slides and peer education

Histology laboratory instruction is moving away from the sole use of the traditional combination of light microscopes and glass slides in favor of virtual microscopy and virtual slides. At the same time, medical curricula are changing so as to reduce scheduled time for basic science instruction as well as focusing on student‐centered learning approaches such as small group active learning and peer‐instruction. It is important that medical schools resist the temptation to respond to this conjunction of events by turning histology into a self‐study activity. This article describes a lymphoid histology laboratory exercise, occurring in a specially equipped Learning Studio housing an entire medical class that utilizes virtual slides in the context of small group active learning and peer instruction. Anat Sci Educ

Calcium-regulated phosphorylation of proteins in the membrane-matrix compartment of the Chlamydomonas flagellum☆

Crosslinking of surface-exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. Previous work has shown that these membrane glycoprotein movements are dependent on a critical concentration of free calcium in the medium and are inhibited reversibly by calcium channel blockers and the protein kinase inhibitors H-7, H-8, and staurosporine. These observations suggest that the flagellum may use a signaling pathway that involves calcium-activated protein phosphorylation to initiate flagellar membrane glycoprotein movements. In order to pursue this hypothesis, we examined the calcium dependence of phosphorylation of flagellar membrane-matrix proteins using an in vitro system containing [gamma-32P]ATP or [35S]ATP gamma S. Using only endogenous enzymes and endogenous substrates found in the membrane-matrix fraction obtained by extraction of flagella with 0.05% Nonidet P-40, we observed both calcium-independent protein phosphorylation and calcium-dependent protein phosphorylation in addition to an active protein dephosphorylation activity. Addition of micromolar free calcium increased the amount of protein phosphorylation severalfold. Calcium-activated protein kinase activity was inhibited by H-7, H-8, and staurosporine, the same protein kinase inhibitors that inhibit the calcium-dependent glycoprotein redistribution in vivo. A small group of polypeptides in the 26-58 kDa range exhibited a dramatic increase in phosphorylation in the presence of 20 microM free calcium. We suggest that Chlamydomonas utilizes the intraflagellar free calcium concentration to regulate the phosphorylation of specific flagellar proteins in the membrane-matrix fraction, one or more of which may be involved in regulating the machinery responsible for flagellar membrane glycoprotein redistribution.

Gliding Motility and Flagellar Glycoprotein Dynamics in Chlamydomonas

Although there are many forms of motility and contractility within eukaryotic cells, most cases of whole cell locomotion can be conveniently divided into one of two classes: (1) movement of cells through a liquid medium due to the propagation of bends along cilia and flagella or (2) movement of cells while in adhesive contact with a solid or semisolid surface. In the first case (swimming of ciliated and flagellated cells), locomotion results from a viscous coupling between the ciliary or flagellar surface and the liquid medium. In the second case (exemplified by amoeboid and fibroblastic movements), cell-surface molecules with specific binding properties mediate the transfer of energy between the cell and the extracellular matrix or solid substrate (Buck and Horwitz, 1987; Burridge et al., 1988; Lackie, 1986).

Protein targeting to flagella of trypanosomatid protozoa

Much exciting research has been done on the co-translational and post-translational targeting ofspecific classes of proteins to membrane-boundedorganelles using targeting sequences or motifslocated within the proteins themselves. In contrast,cilia and flagella have not always been consideredas organelles in the same sense as rough endoplas-mic reticulum, mitochondria or the nucleus; theyhave been seen partly as cell surface specializationsand partly as an extension of the cytoskeleton, bothof which are true. However, despite the fact thatthese organelles are not totally enclosed by a mem-brane, there is good reason to view them as rela-tively ‘closed’ compartments. If this view is correct,one would expect cilia and flagella to utilize specifictargeting and import mechanisms for both mem-brane proteins being delivered via a vesicular trans-port pathway from the Golgi and cytoskeletalproteins that are synthesized on free polyribo-somes. Cilia and flagella can be viewed as tubularextensions of the plasma membrane, ‘plugged’ attheir proximal end by a very complex cytoarchitec-ture (including the basal body and transition zone)tightly associated with the adjacent plasma mem-brane by structures referred to as ciliary necklaces.These basal structures clearly serve as selective bar-riers; for instance, the soluble compartment of ciliaand flagella does not have the same composition asthe general cytoplasm. In terms of comparisonswith other cellular compartments with known tar-geting systems, cilia and flagella can best be com-pared with the nucleus. Entry into and exit from thenucleus does not require passage across a mem-brane but rather occurs through a complex cytoar-chitecture (the nuclear pore) that allows proteinsbelow a certain size to diffuse through an aqueousspace while utilizing a complex mechanism for im-port and export of larger proteins. Cilia and flagellamay need to utilize similar targeting and uptakemechanisms involving targeting sequences ormotifs in the proteins destined for cilia and flagella,along with an active translocation apparatus. Forthe first time, a recent group of papers have re-ported the presence of specific targeting sequencesor motifs in both membrane and cytoskeletal pro-teins destined for flagella of trypanosomatids, afamily of flagellated, parasitic protozoa.Cilia and flagella can be viewed as organellescontaining three ‘domains’: a membrane domain, ahighly structured cytoskeleton (the axoneme) and asoluble compartment. This latter compartment hasbeen variously referred to as the ‘matrix’ compart-ment or ‘flagelloplasm’ or ‘cilioplasm’. The ciliaryor flagellar membrane is a specialized domain ofthe plasma membrane with a protein and lipidcomposition distinct from that of the rest of theplasma membrane (Bloodgood, 1990). A numberof membrane proteins are known to be specificallylocalized to various ciliary or flagellar membranes;among the trypanosomatids, these include specificisoforms of glucose transporters (Snapp andLandfear, 1997), adenylate cyclases (Paindavoine