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Lab 3.1: Smooth Endoplasmic Reticulum, Spine Apparatus, and Endosomes

NEU466G, Dr. Kirk · Class documents

Overview

In this lab you look for smooth endoplasmic reticulum (SER), spine apparatus (SA), and endosomes in 5 additional spines, and stamp each one you find. Each Figure box links a journal figure from the papers listed under References; open them while you work. The EM screenshots on this page show examples from other spines of the series, not from your 5 assigned spines.

Smooth endoplasmic reticulum (SER)

  • SER has a flattened, oval appearance surrounded by a membrane, but this shape can be variable (e.g., on one section the SER can look like a gray smudge, a small black dot, a snake-like black line, or a large vesicle-like lake).
  • SER is connected across serial sections (though sometimes this continuity can be difficult to see).

Figure: snake-like tubule of SER entering a spine

Spacek & Harris, 1997, Fig. 2c. Open the paper.

Section 15 without traces Section 15 with spine d03sp05 in yellow and the SER stamp d03sern05, a yellow cross
SER in spine d03sp05 on section 15, without traces (left) and with d03sp05 and the SER stamp d03sern05 (right). The view is about 1.3 µm across.

Clip: section 13 with spine d03sp05 (yellow) and the SER stamp d03sern05 (yellow cross) in view; the pointer clicks the up arrow four times, through sections 14 to 17.

▶ 8 s · loops

SER in spine d03sp05, sections 13 to 17, stepping up one section at a time.

Spine apparatus (SA)

  • The spine apparatus is a specialized derivative of the SER.
  • SA is typically seen in large mushroom spines but can also appear in the dendritic shaft.
  • SA appears as flat cisternae (arrows) with electron-dense plates between them (wavy arrows); see the figure below.

Figure: spine apparatus, with cisternae (arrows) and dense plates (wavy arrows)

Spacek & Harris, 1997, Fig. 3b. Open the paper.

Section 70 without traces Section 70 with spine d03sp19 in yellow and the SA stamp d03sah19, a red cross
SA in the head of spine d03sp19 on section 70, without traces (left) and with d03sp19 and the SA stamp d03sah19 (right). The view is about 1.6 µm across.

Endosomes

Endosomes are intracellular sorting organelles that are involved with the local recycling, sorting, and degradation of membrane and proteins.

Four major subtypes:

  • Multivesicular body
  • Tubule
  • Smooth vesicle
  • Coated vesicle or pit

Figure: drawing of endosome types in three spines

Cooney et al., 2002. Open the paper.

Endosome criteria

1. Coated vesicles/pits

  • Vesicles: Elliptical with gray interior and clathrin coat (cytoplasmic coat consisting of spoke-like protrusions).
  • Pits: Elliptical, omega-shaped invaginations with gray interiors and clathrin coat.

Figure: clathrin-coated pit, with an inset of a clathrin-coated vesicle

Cooney et al., 2002. Open the paper.

Section 108 without traces Section 108 with spine d03sp29 in yellow and the coated pit stamp d03endo29_cp, an orange octagon
A coated pit on spine d03sp29 on section 108, without traces (left) and with d03sp29 and the stamp d03endo29_cp (right). The view is about 2.3 µm across.

Clip: section 108 in a view 5 µm across; the pointer points at a coated pit and the view zooms in on it, spine d03sp29 (yellow) and the stamp d03endo29_cp (orange) appear, and the view zooms back out.

▶ 9 s · loops

Closing in on the coated pit of spine d03sp29 on section 108.

2. Endosomal tubule

  • Cylindrical compartment with smooth outer membrane + dark, grainy interior.
  • Uniform diameter.
  • May have a clathrin-like protein coat on the end (indicating vesicle budding).

Figure: endosomal tubule

Cooney et al., 2002. Open the paper.

3. Multivesicular body (MVB)

  • Dark, grainy interior with variable number of internal vesicles.

Figure: multivesicular body

Cooney et al., 2002. Open the paper.

4. Vesicles (small/large/amorphous)

  • Small/large smooth vesicles: light gray interiors with smooth outer membrane.
  • Amorphous vesicles: smooth membranes, electron-lucent interiors, and irregular shapes. Often occur in clumps.

Figure: small and large smooth vesicles, and a clump of amorphous vesicles

Cooney et al., 2002. Open the paper.

5 additional spines

To learn to identify these spine resources, you are assigned 5 additional spines:

  • d03p12
  • d03p14
  • d03p37_branched
  • d03p40
  • d03p42

You will search in these spines for SER, SA, and endosomes. These spines have already been fully traced for you (including their c/cfa-traces).

Stamp spine SER and SA

Identify and stamp SER or SA in each of these 5 spines.

Right-click the palette button to open Set Attributes, set Stamp radius (microns) to 0.05, and set the name to d03sa## for SA and d03ser## for SER, with ## corresponding to the spine ##, e.g., SA found in d03p12 would be named d03sa12. For each of the 5 additional spines listed above, stamp any SER and SA you observe within the spine head and spine neck.

Each of the 5 assigned spines will have either SER or SA.

Set Attributes for d03sa12: red, circle, Fill None, Stamp radius 0.05
Set Attributes for d03sa12.

Clip: on section 70 with the Stamp tool, the pointer right-clicks the first palette square, sets the name to d03sa19 and Stamp radius (microns) to 0.05, clicks OK, and clicks inside spine d03sp19 to place the red stamp d03sa19 on the SA.

▶ 12 s · loops

Set the name and the radius, click OK, then click on the SA. Shown in spine d03sp19, not one of your 5 spines.

Stamp spine endosomes

Look for endosomes in each of these 5 spines.

In spines in which endosomes have been identified, place a stamp on the endosomal object.

Hint

You are likely to find a coated pit in these 5 spines. There may be additional endosomal structures that need a second stamp in these spines.

Right-click the palette button to open Set Attributes, set Stamp radius (microns) to 0.05, and set the name to d03endo##_cp (with cp for coated pit and ## corresponding to spine ##). All but one of these spines has at least one endosome.

Set Attributes for d03endo12_cp: orange, circle, Fill None, Stamp radius 0.05
Set Attributes for d03endo12_cp.
The Object List filtered by the group SER, SA, Endo, with columns Name, Start, End, Count and Groups, rows d03endo29_cp to d03sern05
The Object List filtered by the group "SER, SA, Endo", with the first and last section of each stamp.

Extra credit

+0.25 pts extra credit on your lab report if you can correctly identify all coated pits in your spines.

References