Autophagy and turnover of photoreceptor membrane and rhodopsin in Drosophila

SLU Biology Departmental seminar

March 6, 2009

(PodCast of this seminar here)


Here are the last 3 Biology Department seminars I have delivered:


Microscopy of Drosophilas 6 rhodopsins, Fall, 2002


Vision in the ultraviolet, Fall, 2005

This was later recorded as a PodCast when it was given as a lecture in a physiology class


Why is there lutein in your vitamin pill and what does that have to do with Drosophila vision? Fall, 2007

This was later recorded as a PodCast when it was given as a lecture in a physiology class


Also I presented a short lecture for SLUs Tribeta (biology club) February 2009

Drosophila as a model for studies of vision PodCast


In September 2009, I gave a lecture on vitamin A for ³current research in vitamins (Nutrition and Dietetics) (PodCast)


In February 2010, I gave a journal club at ³Neuro at Noon² (pdf) (PodCast)


Marching Bands


Any of the thousands of people who are at least as interested in the vertebrate visual system as I am know that marching bands refers to the migration of label and the turnover of the rod outer segment.


For this work, Richard Young was awarded the Friedenwald Award at ARVO (the Association for Research in Vision and Ophthalmology) in 1976


The first slide I am showing is from Richard W. Young, Visual cells, Scientific American, October 1970,  80-91


This material is also in the text I use for my Neuroscience class - Purves et al., Neuroscience, Sinauer Associates, Inc, New York, 2008 (fourth edition) [next slide]


RPE – phagolysosomal system


RPE=retinal pigment epithelium. These cells phagocytose the debris of the rods.


An SEM I prepared shows rod tips with RPE partially stripped off.


Lipofuscin is an aging pigment that fluoresces yellow to blue excitation. It accumulates with age (since RPE cells are post-mitotic) as shown in this preparation from an 82 year old donor.


My contribution is in a Science paper, as our envious chemistry and biochemistry colleagues were overheard saying:

Stark and Eldred got a Science paper out of the notorious insensitivity of spectrofluorometers to short wavelengths


Light and dark, circadian rhythms


In the invertebrate, there are differences in the rhabdom (the photoreceptive organelle) with light vs dark adaptation (next 3 slides):


RHWhite and ELord, 1975, Diminution and enlargement of the mosquito rhabdom in light and darkness, J.Gen.Physiol.65, 583-598


DRNassel and THWaterman, 1979, Massive diurnally modulated photoreceptor membrane turnover in crab light and dark adaptation, J.Comp.Physiol.A 131, 205-216


SCChamberlain and RBBarlow, Jr., 1984, Transient membrane shedding in Limulus photoreceptors: control mechanisms under natural lighting, JNeurosci. 4, 2792-2810


My own work (on circadian rhythm)


My whiz-kid J. Scott Christianson was helpful in many aspects of this work. Now he is founder and in charge of Kaleidoscope Videoconferencing


Flies are stuck down on a microscope slide

(Hyperlink [slide] is from Stark and Thomas 2004)


My microscope can be used to make photometric measurements


Rhodopsin decreased after light onset and increased in the dark; this cycle persisted in constant darkness


My coworker De-Mao Chen did magic with electrophysiology


An electrode is poked into the eye

(Slide is from my Drosophila ERG presentation in the Drosophila neurobiology course at Cold Spring Harbor)


The light-dark differences (and the endogenous rhythm) were more obvious in the ERG (electroretinogram) sensitivity


Cytoarchitecture of turnover


Out of a considerable body of work, I thought one of the most interesting findings was Eguchi & Watermanıs (1976). Based on which half membrane the protein is on (protoplasmic vs exoplasmic half leaflet [P face vs E face]), it appears that vesicles from the rhabdom are re-endocytosed into a multivesicular body (MVB)


EEguchi and THWaterman, 1976, Freeze-etch and histochemical evidence for cycling in crayfish photoreceptor membranes, Cell Tiss. Res. 169, 419-434


Electron microscopy (my turnover studies)


I thank my mentor for electron microscopy, Stanley D. Carlson, now emeritus, Department of Entomology, University of Wisconsin – Madison


I thank my electron microscopy technician Randy Sapp. He won a staff recognition award for this work. Eventually, he became an  optometrist, practicing in St. Joseph, MO


A series of papers started with Stark, Sapp and Schilly 1988


Coated pits form from the rhabdomeres


I must thank a colleague, Lynnette Feeney-Burns, now retired, for useful discussions and for  introducing me to the concept of autophagy


Coated pits also form from the plasmalemma


Coated pits fail to pinch off in shibire, a temperature sensitive mutant of dynamin,  at the restrictive temperature


The next slide is the cartoon from the neuroscience text I use (Purves et al.) about the role of dynamin in vesicle recycling


Multivesicular bodies (MVBs), often in the company of primary lysosomes, were observed

Note, this was stained for acid phosphatase, but in an acid phosphatase negative Drosophila mutant


Acid phosphatase staining (except in an acid phosphatase deficient mutant, above) suggested that MVB recycling started with digestion by lysosomes


norpA (the no receptor mutant, a phospholipase C defect) has extensive areas of plasmalemma that look like coated pits (zippers)


ora (outer rhabdomeres absent, a nonsense mutant of rhodopsin) also elaborates such massive membrane


Rhodopsin synthesis


Rough Endoplasmic Reticulum (near Golgi apparatus) is shown as a reminder that this entire line of work relates to general cell biological issues such as protein synthesis and trafficking


Vitamin A deprivation:


Rhabdomeres are intact but lack rhodopsin


Nature paper


Rhabdomeres are smaller


Vitamin A replacement therapy


Opsin immunogold decoration is high as carrot juice synchronizes a massive de novo synthesis and export


Circles (membrane vesicles) may be the export vehicle


Collaboration with Joel Eissenberg, Professor of Biochemistry, SLU


Pilot studies Summer 2008

(Prof. Eissenbergıs seminar is March 20)

Sabbatical spring semester 2010


The Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA) proteins


Flies are stuck down on a microscope slide (I am showing this figure again)

(Hyperlink [slide] is from Stark and Thomas 2004)


Diagram Optics (deep pseudopupil)


Optical method shows rhabdomeres degenerating by 8 days

Deep pseudopupil shows optics are abnormal from the outset


Question – Answer period


A question about why Drosophila? allowed discussion of advantages of Drosophila as well as strategic techniques


Prof. Eisenberg commented that, since a mutant was lethal, tissue (eye) – specific knockdown was the ideal Drosophila strategy.


Jan Barber asked about homologies of rhodopsin and the answer I gave referred to the evolution of G protein coupled receptors such as I gave in the vision lecture of my neuro course


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